EP0498849A1 - Means for detecting highly proliferative cells - Google Patents

Means for detecting highly proliferative cells

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Publication number
EP0498849A1
EP0498849A1 EP19900917289 EP90917289A EP0498849A1 EP 0498849 A1 EP0498849 A1 EP 0498849A1 EP 19900917289 EP19900917289 EP 19900917289 EP 90917289 A EP90917289 A EP 90917289A EP 0498849 A1 EP0498849 A1 EP 0498849A1
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EP
European Patent Office
Prior art keywords
ndp
kinases
sequence
nucleotides
biological sample
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.)
Withdrawn
Application number
EP19900917289
Other languages
German (de)
French (fr)
Inventor
Marie-Lise Lacombe
Valérie WALLET
Michel Veron
Michèle Mock
Octavian Barzu
Robert Sarfati
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut Pasteur de Lille
Original Assignee
Institut Pasteur de Lille
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Filing date
Publication date
Priority claimed from FR8914328A external-priority patent/FR2653781B1/en
Priority claimed from FR9004754A external-priority patent/FR2660933A1/en
Application filed by Institut Pasteur de Lille filed Critical Institut Pasteur de Lille
Publication of EP0498849A1 publication Critical patent/EP0498849A1/en
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the subject of the invention is a method for the detection of highly proliferative cells such as tumor cells, more particularly malignant tumors, and a kit allowing the implementation of such a method.
  • the invention therefore aims to provide a method of detecting highly proliferative cells, in particular, it aims to provide a method for demonstrating a potentially progressive tumor state by measuring the activity or the level of particular enzymes.
  • the invention aims to provide a diagnostic kit for the implementation of this method.
  • the method according to the invention for the detection of highly proliferative cells is characterized in that it comprises the in vitro measurement of the activity or of the amount of NDP kinases of a biological sample.
  • NDP kinases nucleoside diphosphate kinases
  • PARKS & AGARWAL (1973) The Enzymes, ed. Boyer, PD (Acadisputedmie Press, NY), Vol. 8, pp. 307-334).
  • These enzymes catalyze the transfer of the 7- phosphate group from a donor triphosphonucleoside to an acceptor nucleoside diphosphate by a ping-pong mechanism comprising a stable phosphorylated intermediate as shown below:
  • and N 2 are ribo and deoxyribonucleosides and EAP, the phosphorylated intermediate
  • nucleotide substrates Their specificity with respect to nucleotide substrates is quite broad since they can use either purine and pyrimidine derivatives as well as ribose and deoxyribose derivatives. They form polymers made up of one or two types of monomers most often associated in hexamers.
  • the subject of the invention is a method of in vitro detection of malignant tumors, or even of progression from a benign tumor to a malignant tumor (presence of metastases), carried out from a biological sample capable of presenting such a tumor, in particular a tissue sample taken from a patient, characterized in that the possible increase in the production of NDP kinases in this biological sample is measured, compared with the NDP-kinases normally produced in a healthy reference biological sample, same nature as the biological sample studied above.
  • the possible increase in the production of NDP kinases is detected by measuring the enzymatic activity NDP kinase in the biological sample studied.
  • the measurement of the activity of NDP kinases is advantageously carried out by bringing the biological sample studied into contact with natural nucleotides as substrates, and more particularly nucleoside diphosphates and triphosphates, or analogs of synthesis of these nucleotides, and by highlighting the possible products of the reaction between these nucleotides, catalyzed by NDP kinases, by coupled enzymatic systems allowing the synthesis of products detectable by color and / or spectrophotometry.
  • hexokinase-glucose-6-phosphate dehydrogenase system linking the formation by NDP kinase of ATP from ITP (inosine triphosphate) and ADP, to the formation of NADPH + H + , products detectable spectrophotometrically directly at 340 nm or in the visible spectrum after the reaction with phenazine methosulfate and tetrazoliu nitroblue (CHENG et al, 1971, Biochemistry 10, 2139).
  • the enzymatic activity is measured by a coupled enzymatic system comprising the pyruvate kinase which uses the ADP formed by the NDP kinase from ATP and DTDP.
  • ADP and phosphoenolpyruvate give ATP and pyruvate.
  • the latter reacts with phenylhydrazine which transforms into phenylhydrazone, a compound detectable at 450 nm.
  • This assay can be performed on an ELISA plate.
  • NDP kinase activity can also be followed using natural nucleotides or radioactive synthetic analogs and techniques for separation of reaction products by chromatography (PARKS & AGARWAL, 1973, in The enzymes (BOYER PD Ed), 8 , 307, Academy Press, NY).
  • the possible increase in the production of NDP kinases is detected by measuring the amount of NDP kinases in the biological sample studied.
  • the quantity of NDP kinases is measured by labeling the phosphorylated intermediate resulting from the interaction of the nucleotide triphosphate substrate with the enzyme, the labeling of the phosphorylated intermediate being carried out using analogues such as derivatives. 7 - thiophosphates of ATP or GTP which present the advantage of not being able to be hydrolyzed by phosphatases.
  • the amount of NDP kinases present is measured using polyclonal anti-NDP kinase antibodies or monoclonal, and the possible immunological complexes formed between the NDP kinases and the abovementioned antibodies are highlighted. This detection can be carried out on a histological section or after electrophoresis of the sample and transfer of WESTERN, or by any other appropriate means.
  • the anti-NDP kinase antibodies mentioned above are chosen from the antibodies capable of recognizing any NDP kinase, and more particularly from those directed against all or part of the A and / or B subunits of the NDP kinases of human erythrocytes (Presecan, E et al. (1989) FEBS lett. 250, 629-632), or those directed against all or part of the proteins described by Rosengard, AM et al. in NATURE 342, 177-180 (1989) and which it has been shown to correspond to NDP kinases (allet et al., J. Nat. Cancer Inst. (1990) 82, 1199-1202), or those raised against all or part of the NDP kinase shown in Figure 1, and which is described below.
  • the most common developing systems include chemical, physical and / or enzymatic and / or chemiluminescent markers detectable when the antigen-antibody type reaction has occurred. Mention will be made of colorimetric systems coupled with enzymes, fluorescent or even radiolabelled systems.
  • the present invention relates to polyclonal or monoclonal anti-NDP kinase antibodies capable of recognizing all or part of the protein with NDP kinase activity (and to bind immunologically with this protein) as obtained by cloning the gene coding for NDP kinase from Dictyostelium discoideum and purification of the synthesized protein.
  • This protein constitutes one of the objects of the invention as a new product.
  • the protein with NDP kinase activity of the invention is further characterized in that it is strongly basic with a calculated pi of 9.15, in that it contains a hydrophobic fragment (residues 64 to 84), and in that 'It is particularly stable. Its basic properties allow it to be easily purified.
  • This protein therefore constitutes an industrial source of NDP kinase of great interest compared to NDP kinases currently obtained from extracts of tissues or organs and the purification of which requires the implementation of expensive techniques.
  • the invention also relates to a nucleotide sequence consisting of at least part of a sequence coding for a protein with NDP kinase activity as expressed by D. discoideum.
  • This sequence is capable of hybridizing with genes coding for a protein with NDP kinase activity.
  • the percentage of homology with regard to the sequences which hybridize is from 70 to 90%.
  • the conditions for the hybridizations mentioned in connection with the new nucleotide sequences are relatively low stringency conditions.
  • 30% of formamide, 6xSSC (sodium citrate, NaCl), 0.12 M of sodium pyrophosphate, 0.5% of SDS and 250 ⁇ g of herring sperm DNA are used at 30 ⁇ C. washes are performed in the same medium at 37 ° C and according to the importance of the background noise, by gradually decreasing the concentration of SSC and increasing the wash temperature by 5 ° C 5 ° C.
  • the invention also relates to a recombinant sequence comprising one of the sequences defined above, optionally associated with a promoter capable of controlling the transcription of the DNA sequence coding for transcription termination sequences and translation and secretion.
  • the nucleotide sequence of the invention is capable of hybridizing with a probe formed from the sequence having the sequence of nucleotides shown in FIG. 1 or the sequence of nucleotides complementary to this sequence .
  • sequences of the invention are DNA or RNA sequences.
  • nucleotide sequence hybridizable with that of the sequence of FIG. 1, as obtained by reverse enzymatic transcription of the corresponding RNA, or also by chemical synthesis, is part of the invention.
  • the cloning of the nucleotide sequence coding for a protein with NDP kinase activity comprises the isolation of clones of an expression library constructed in a vector, of the type of those expressing a protein interacting specifically with a compound such as [ 5 S] GTP 7 S, triphosphate donor.
  • a vector is constructed by inserting a nucleotide fragment containing the coding sequence.
  • the plasmid pNDK is constructed, for example, to express the protein with NDP kinase activity in a bacterium such as E. coli.
  • a bacterium such as E. coli.
  • the HaelII - ElickRI restriction fragment of the isolated clone is inserted, this fragment having been advantageously treated beforehand to obtain blunt ends.
  • the insertion is carried out in the HincII site of the plasmid pTZ18R.
  • the resulting recombinant plasmid is introduced, according to usual techniques, into the bacterium for the purpose of expression of the enzyme under the control of the lac Z promoter.
  • IPTG inducing agent
  • the enzyme recovered is subjected to at least two purification steps on a chromatography column.
  • a homogeneous enzyme preparation is obtained by contacting a centrifugation supernatant of a suspension of bacteria previously broken by sonication, obtained at the end of the above process, with a dextran derivative such as DEAE-Sephacel ®, (at pH 8.5, the protein NDP kinase activity is not retained in the DEAE resin and is eluted in the void volume), the eluate is then collected chro atographié on a resin column such as Affigel-Blue ® , and eluted with 0.7M NaCl.
  • a dextran derivative such as DEAE-Sephacel ®
  • the applications of the products according to the invention also include the development, from fragments of the nucleotide sequence of FIG. 1, of RNA, mRNA or DNA probes, for the detection of similar sequences in other organisms or tissues.
  • This elaboration includes, in particular, the denaturation of the double-stranded sequences to obtain a single-stranded sequence usable as a probe.
  • the invention therefore relates to detection probes characterized in that they comprise at least part of a nucleotide sequence defined above, of RNA or DNA, more especially a fragment of a sequence coding for a protein with NDP kinase activity.
  • the fragment used as probe contains a sufficient number of nucleotides to obtain the specificity and to form a stable hybrid.
  • Appropriate probes are advantageously marked with a radioactive element (hot probes) or any other non-radioactive group (cold probes) allowing recognition of the probe in a hybrid state with the preparation containing the DNA to be studied.
  • a radioactive element hot probes
  • cold probes any other non-radioactive group
  • cDNA probes specific for NDP kinases of the type described above are used, in particular probes capable of hybridizing with the corresponding sequences of the mRNAs coding for NDP kinases, an increase in the level of mRNA may be linked to an increase in the amount of NDP kinase proteins reflecting the tumor state of the sample.
  • the activity or the quantity of NDP kinases is measured on a biological sample originating from a patient.
  • an activity comparison is more particularly carried out between a biological sample containing highly proliferative cells and a sample containing normal corresponding cells.
  • biological sample any sample taken from the patient, whether it is a solid tumor or a biological fluid such as blood, serum, cerebrospinal fluid, pleural fluid, urine. , sputum, samples obtained by tubing, bronchial aspiration, or puncture, or circulating cells.
  • a biological fluid such as blood, serum, cerebrospinal fluid, pleural fluid, urine. , sputum, samples obtained by tubing, bronchial aspiration, or puncture, or circulating cells.
  • the biological sample is used in whole, crushed, sectioned, fractional form, in particular in the form of sub ⁇ cell fractions or, where appropriate, in the form of cell culture.
  • the reagents necessary for demonstrating the activity or the amount of NDP kinases can be in a liquid medium or incorporated into a support material, for example gels or support filters.
  • the invention also relates to kits for the in vitro detection of malignant tumors.
  • kits are characterized in that they contain the necessary reagents as defined above for demonstrating the possible increase in the production of NDP kinases when these reagents are brought into contact with a biological sample in appropriate form.
  • the detection kits of the invention have the advantage of being able to be used directly on clinical samples and allow results to be obtained in a very rapid time.
  • the invention relates to detection kits characterized in that they contain:
  • nucleoside diphosphates and triphosphates or analogs of synthesis of these nucleotides optionally radioactive labeled, or in the form of 7- thiophosphate derivatives of ATP or of GTP,
  • a suitable reaction medium for the enzymatic reaction between nucleotides and NDP kinases coupled enzymatic systems allowing the synthesis of products detectable by colorimetry and / or spectrophotometry, to 7- thiophosphates derivatives of ATP or of GTP allowing labeling of the phosphorylated intermediate, or of the kits containing: polyclonal or monoclonal anti-NDP kinase antibodies, where appropriate labeled, in particular in a radioactive or enzymatic manner,
  • kits suitable for the detection of the immunological complexes formed between the abovementioned antibodies and the NDP kinases, or also kits comprising:
  • the invention also relates to pharmaceutical compositions comprising all or part of one (or more) protein with NDP kinase activity, in particular that obtained by cloning the gene coding for NDP kinase from Dictyos e1ium discoideum and purification of the synthesized protein, or alternatively that corresponding to subunits A and / or B of human erythrocytes (cf. Presecan et al., and Rosengard et al. mentioned above) in association with a physiologically acceptable vehicle.
  • the invention relates more particularly to pharmaceutical compositions comprising all or part of the amino acid sequence shown in FIG. 1 in association with a physiologically acceptable vehicle.
  • a subject of the invention is also pharmaceutical compositions comprising anti-NDP kinase polyclonal or monoclonal antibodies as designated above, in association with a physiologically acceptable vehicle.
  • the invention also relates to pharmaceutical compositions comprising nucleotide sequences as defined above, and more particularly antisense nucleotide sequences complementary to all or part of the nucleotide sequence coding for a protein with NDP kinase activity, in particular of the nucleotide sequence shown in Figure 1, in association with a physiologically acceptable vehicle.
  • compositions of the invention are more particularly intended for treatment of tumors and in particular the prevention of the progression from a benign tumor to a malignant tumor.
  • These pharmaceutical compositions have the advantage of inhibiting NDP kinase activity.
  • the invention also relates to any nucleic acid sequence included in one of the nucleic acid sequences defined above, or capable of hybridizing with one of these sequences under the hybridization conditions defined above, and being usable as as a nucleic acid primer for the gene amplification of one of the nucleic acid sequences of the invention.
  • nucleic acid primers of the invention consist of a succession of approximately 10 to 30 nucleotides.
  • Gene amplification techniques are a considerable adjunct to the development of particularly sensitive in vitro diagnostic methods.
  • PCR Poly erase Chain Reaction
  • the technique called "Q / 3replicase” described in Biotechnology, vol.6, page 1197 (October 1988) and that using a RNA polymerase (T7RNA polymerase) described in the international patent application No. WO89 / 01050.
  • T7RNA polymerase RNA polymerase
  • the polyclonal antibodies of the invention are obtained by immunization of an animal with the polypeptides of the invention, followed by the recovery of the antibodies formed.
  • the monoclonal antibodies of the invention are produced by any hybridoma capable of being formed, by conventional methods, from the ⁇ plenic cells of an animal, in particular mouse or rat, immunized against one of the purified polypeptides of l invention, on the one hand and cells of a cell line of a suitable myeloma on the other hand, and to be selected, by its capacity to produce monoclonal antibodies recognizing the polypeptide initially used for the animal immunization.
  • Another method for preparing the nucleotide sequences of the invention comprises the following steps:
  • the subject of the invention is also any recombinant nucleic acid containing at least one nucleic sequence of the invention, inserted into a nucleic acid heterologous with respect to said nucleic sequence.
  • the invention relates more particularly to a recombinant nucleic acid as defined above, in which the nucleic sequence of the invention is preceded by a promoter (in particular an inducible promoter) under the control of which the transcription of said sequence is capable of be performed and, where appropriate, followed by a sequence encoding transcription termination signals.
  • a promoter in particular an inducible promoter
  • the invention relates to any recombinant vector, used in particular for the cloning of a nucleic sequence of the invention, and / or the expression of the polypeptide encoded by this sequence, and characterized in that it contains a recombinant nucleic acid, as defined above, at one of its sites not essential for its replication.
  • the subject of the invention is also a process for the preparation of a polypeptide of the invention, by transformation of a cellular host using a recombinant vector of the above-mentioned type, followed by the cultivation of the cell host thus transformed, and recovery of the polypeptide from the culture medium.
  • the invention relates to any cellular host transformed by a recombinant vector as defined above, and comprising the regulatory elements allowing the expression of the nucleic sequence coding for a polypeptide according to the invention.
  • the peptides according to the invention can be prepared by conventional techniques, in the field of peptide synthesis. This synthesis can be carried out in homogeneous solution or in solid phase.
  • tissue fragments obtained frozen in liquid nitrogen are quickly thawed in 10 mM Tris-HCl buffer, pH 7.5 maintained at 4 ° C., containing 1 mM EDTA, 20% glycerol and inhibitors of proteases (PMSF (phenyl methyl sulfonyl fluoride) 1 mM, 1 ⁇ g / ml of antipain and 15 ⁇ g / ml of benzamidine) at a rate of approximately 5 ml of buffer per gram of tissue.
  • the fabrics are finely cut and then ground with Polytron® (3 times 20 seconds at maximum power). The ground material is then centrifuged at 4 "C., 10 min. At 20,000 g in a refrigerated centrifuge. The supernatant is used immediately or stored in aliquots at -80 ° C. protein concentration thereof is measured by the method of BRADFORD (1976, Anal. Biochem., 72, 248). Determination of NDP kinase activity
  • the enzymatic activity is measured by an enzyme system coupled with pyruvate kinase-lactate dehydrogenase (AGARWAL et al, 1978, Meth. Enzy ol., 51, 376) which links the formation of ADP by the NDP kinase (s) ) to a consumption of NADH detected spectrophotometrically at 340 n.
  • AGARWAL et al, 1978, Meth. Enzy ol., 51, 376 which links the formation of ADP by the NDP kinase (s) ) to a consumption of NADH detected spectrophotometrically at 340 n.
  • the reactions are as follows:
  • PEP phosphoenolpyruvate
  • PK pyruvate kinase
  • LDH lactate dehydrogenase
  • dTDP deoxythymidine diphosphate
  • dTTP deoxythymidinetriphosphate
  • the reaction takes place at 30 ° C in a total volume of 500 ⁇ l of 50 mM Tris-HCl, pH 7.4, containing 0.5 mM phosphoéololpyruvate, 0.5 mM ATP, MgCl 2 6 mM, 100 ⁇ g / ml of NADH, 50 mM KC1, 0.2 mM dTDP, 1.5 U of pyruvate kinase and lactate dehydrogenase and the quantity of tis ⁇ ular extract supernatant (1 to 10 ⁇ l) necessary for l '' obtaining a speed directly proportional to the amount of extract.
  • a unit of enzyme catalyzes the formation of one ⁇ mole of ADP per minute.
  • these measurements were carried out at 30 ° C. in a 0.5 ml reaction medium consisting of 50 mM tris HCl, pH 7.4, 50 mM KCl, 6 mM MgCl 2 , 1 mM phosphoenolpyruvate, 0.1 mg / ml NADH, 0.5 mM ATP, 0.1 mM dTDP, 2 units of pyruvate kinase and 2.5 units of lactate dehydrogenase.
  • NDP kinase activity of extracts from several solid breast tumors was compared with that of different normal tissues by the pyruvate kinase-lactate dehydrogenase method.
  • NDP kinase activity of extracts from normal lymph nodes B7, H2), normal muscles (D11, A2), normal skin (A3), normal breasts (A5, B8, J6, K8), normal thyroid (E12), benign breast tumors (Gl, O5, N4, Q7, T10), malignant breast tumors (A1, B6, F13, H3, J7, K9, V12 , P6), malignant neoplasms of lymph nodes (A4, U11), malignant neoplasms of the colon (L2, R8), and malignant neoplasms of the cervix (M13).
  • the increase in the NDP kinase enzyme activity corresponds to an increase in the quantity of the enzyme (and more particularly of the A sub-unit of the NDP kinase of human erythrocytes), as has been evaluated by WESTERN transfer , using antibodies specific for NDP kinase from human erythrocytes. These results are confirmed on tissue section by specific labeling of tumor cells. NDP kinase activity has been measured in extracts from many normal tissues or from malignant or benign tumors. A remarkable and specific increase in the production of NDP kinase has been observed in malignant tumors (see Figure 2). The NDP kinase activity of normal tissues varies between 0.1 and 0.3 units (U) per mg of protein.
  • NDP kinase activity is clearly higher in the extracts of malignant tumors, while the values measured in the extracts of benign tumors are similar to those measured in normal tissues. Most of the values found in malignant tumors range from 0.5 to 0.9 U / mg protein. The highest activity was measured in a breast tumor (non-infiltrating intraductal carcimone (P6)) and corresponds to 1.93 U / mg of protein. The lowest value, 0.25 U / mg protein, was found in colon adenocarcimone (R8).
  • the measurement of NDP kinase activity therefore constitutes a criterion for evaluating the potential progressive tumor state of a tissue sample.
  • the invention thus provides a simple test for screening for potentially progressive tumor states.

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Abstract

L'invention a pour objet une méthode pour la détection de cellules fortement prolifératives telles que les cellules tumorales et un kit dans lequel on mesure l'activité ou la quantité de NDP kinases.The subject of the invention is a method for the detection of highly proliferative cells such as tumor cells and a kit in which the activity or the quantity of NDP kinases is measured.

Description

MOYENS POUR LA DETECTION DE CELLULES HAUTEMENT MEANS FOR DETECTION OF HIGHLY CELLS
PROLIFERATIVES L'invention a pour objet une méthode pour la détection de cellules fortement prolitératives telles les cellules tumorales, plus particulièrement les tumeurs malignes, et un kit permettant la mise en oeuvre d'une telle méthode.PROLIFERATIVES The subject of the invention is a method for the detection of highly proliferative cells such as tumor cells, more particularly malignant tumors, and a kit allowing the implementation of such a method.
A l'heure actuelle, la détection de tumeurs fait appel le plus souvent à des examens histologiques des cellules transformées qui nécessitent un personnel hautement qualifié pour apprécier les résultats. De plus, peu de marqueurs fiables de cellules tumorales sont actuellement disponibles.At present, the detection of tumors most often calls for histological examinations of the transformed cells which require highly qualified personnel to assess the results. In addition, few reliable markers of tumor cells are currently available.
L'étude de tumeurs par les inventeurs, notamment de tumeurs du sein, du colon, du col utérin, de métastases ganglionnaires, de mélanome, a montré une augmentation importante de 1'activité et du taux d'une enzyme intervenant dans la synthèse des nucléotides (la nucléoside diphosphate kinase) .The study of tumors by the inventors, in particular tumors of the breast, colon, cervix, lymph node metastases, melanoma, has shown a significant increase in the activity and the level of an enzyme involved in the synthesis of nucleotides (the nucleoside diphosphate kinase).
La mesure de 1•activité ou du taux de cette enzyme a pu être correlée de manière reproductible à l'état tumoral des échantillons biologiques testés.The measurement of the activity or of the level of this enzyme could be reproducibly correlated with the tumor state of the biological samples tested.
L'invention a donc pour but de fournir une méthode de détection de cellules hautement prolifératives, en particulier, elle vise à fournir une méthode pour mettre en évidence un état tumoral potentiellement évolutif en mesurant l'activité ou le taux d'enzymes particulières.The invention therefore aims to provide a method of detecting highly proliferative cells, in particular, it aims to provide a method for demonstrating a potentially progressive tumor state by measuring the activity or the level of particular enzymes.
Selon un autre aspect, l'invention vise à fournir un kit de diagnostic pour la mise en oeuvre de cette méthode.According to another aspect, the invention aims to provide a diagnostic kit for the implementation of this method.
La méthode, selon l'invention, pour la détection de cellules fortement prolifératives, est caractérisée en ce qu'elle comprend la mesure in vitro de l'activité ou de la quantité de NDP kinases d'un échantillon biologique. On sait que les nucleosides diphosphate kinases (NDP kinases; EC 2.7.4.6.) jouent un rôle majeur dans la synthèse des nucleosides triphosphates (PARKS & AGARWAL (1973), The Enzymes, éd. Boyer, P.D. (Académie Press,NY), Vol. 8, pp. 307-334). Ces enzymes catalysent le transfert du groupement 7-phosphate d'un triphosphonucléoside donneur sur un nucléoside diphosphate accepteur par un mécanisme ping-pong comportant un intermédiaire phosphorylé stable comme représenté ci-après :The method according to the invention for the detection of highly proliferative cells is characterized in that it comprises the in vitro measurement of the activity or of the amount of NDP kinases of a biological sample. We know that nucleoside diphosphate kinases (NDP kinases; EC 2.7.4.6.) Play a major role in the synthesis of nucleoside triphosphates (PARKS & AGARWAL (1973), The Enzymes, ed. Boyer, PD (Académie Press, NY), Vol. 8, pp. 307-334). These enzymes catalyze the transfer of the 7- phosphate group from a donor triphosphonucleoside to an acceptor nucleoside diphosphate by a ping-pong mechanism comprising a stable phosphorylated intermediate as shown below:
N?DP + EΛ-*P a N,TP + E N ? DP + EΛ- * P a N, TP + E
N,TP + N2DP ≈ NiDP + N2TPN, TP + N 2 DP ≈ NiDP + N 2 TP
(N.| et N2 sont des ribo et déoxyribonucléosides et EAP, 1'intermédiaire phosphorylé)(N. | and N 2 are ribo and deoxyribonucleosides and EAP, the phosphorylated intermediate)
Leur spécificité vis-à-vis des substrats nucléotidiques est assez large puisqu'elles peuvent utiliser indifféremment les dérivés puriques et pyrimidiques et aussi bien les dérivés riboses que déoxyriboses. Elles forment des polymères constitués d'un seul ou de deux types de monomères associés le plus souvent en hexamères.Their specificity with respect to nucleotide substrates is quite broad since they can use either purine and pyrimidine derivatives as well as ribose and deoxyribose derivatives. They form polymers made up of one or two types of monomers most often associated in hexamers.
Les inventeurs ont observé que les variations d'activité ou de taux de NDP kinases (variations allant dans le sens d'une augmentation de l'activité ou du taux de NDP kinases par rapport à cette activité ou ce taux chez un individu normal) traduisent un état tumoral potentiellement évolutif. L'invention a pour objet une méthode de détection in vitro de tumeurs malignes, voire d'évolution d'une tumeur bénigne vers une tumeur maligne (présence de métastases) , réalisée à partir d'un échantillon biologique susceptible de présenter une telle tumeur , notamment un prélèvement de tissu effectué chez un patient , caractérisée en ce que l'on mesure l'éventuel accroissement de la production de NDP kinases dans cet échantillon biologique, par rapport aux NDP-kinases normalement produites dans un échantillon biologique sain de référence, de même nature que l'échantillon biologique étudié sus-mentionné.The inventors have observed that the variations in activity or in the level of NDP kinases (variations going in the direction of an increase in the activity or in the level of NDP kinases in relation to this activity or this rate in a normal individual) reflect a potentially progressive tumor state. The subject of the invention is a method of in vitro detection of malignant tumors, or even of progression from a benign tumor to a malignant tumor (presence of metastases), carried out from a biological sample capable of presenting such a tumor, in particular a tissue sample taken from a patient, characterized in that the possible increase in the production of NDP kinases in this biological sample is measured, compared with the NDP-kinases normally produced in a healthy reference biological sample, same nature as the biological sample studied above.
Selon un mode avantageux de réalisation de la méthode de détection de 1'invention,1'éventuel accroissement de la production de NDP kinases est détecté en mesurant 1'activité enzymatique NDP kinase dans l'échantillon biologique étudié.According to an advantageous embodiment of the detection method of the invention, the possible increase in the production of NDP kinases is detected by measuring the enzymatic activity NDP kinase in the biological sample studied.
La mesure de l'activité des NDP kinases est réalisée avantageusement en mettant en présence l'échantillon biologique étudié avec des nucléotides naturels en tant que substrats, et plus particulièrement des nucleosides diphosphates et triphosphates, ou des analogues de synthèse de ces nucléotides, et en mettant en évidence les éventuels produits de la réaction entre ces nucléotides, catalysée par les NDP kinases, par des systèmes enzymatiques couplés permettant la synthèse de produits détectables par colori étrie et/ou spectrophotométrie.The measurement of the activity of NDP kinases is advantageously carried out by bringing the biological sample studied into contact with natural nucleotides as substrates, and more particularly nucleoside diphosphates and triphosphates, or analogs of synthesis of these nucleotides, and by highlighting the possible products of the reaction between these nucleotides, catalyzed by NDP kinases, by coupled enzymatic systems allowing the synthesis of products detectable by color and / or spectrophotometry.
Ainsi, on utilise par exemple un système hexoki- nase-glucose-6-phosphate déshydrogénase, reliant la formation par la NDP kinase d'ATP à partir d'ITP (inosine triphosphate) et d'ADP, à la formation de NADPH+H+, produits détectables spectrophotométriquement directement à 340 nm ou dans le spectre visible après la réaction avec la phénazine methosulfate et le nitrobleu de tétrazoliu (CHENG et al, 1971, Biochemistry 10, 2139) .Thus, for example, a hexokinase-glucose-6-phosphate dehydrogenase system is used, linking the formation by NDP kinase of ATP from ITP (inosine triphosphate) and ADP, to the formation of NADPH + H + , products detectable spectrophotometrically directly at 340 nm or in the visible spectrum after the reaction with phenazine methosulfate and tetrazoliu nitroblue (CHENG et al, 1971, Biochemistry 10, 2139).
D'autres systèmes enzymatiques couplés utilisables dans le cadre de la mesure de l'activité NDP kinase, sont plus particulièrement décrits dans Parks et Agar al sus¬ mentionné, et Kezdi et al, Anal. Biochem. , 361-364 (1976) .Other coupled enzymatic systems usable within the framework of the measurement of the NDP kinase activity, are more particularly described in Parks and Agar al above-mentioned, and Kezdi et al, Anal. Biochem. , 361-364 (1976).
Dans l'article de Kezdi et al, sus mentionné, 1'activité enzymatique est mesurée par un système enzymatique couplé comportant la pyruvate kinase qui utilise l'ADP formé par la NDP kinase à partir d'ATP et de DTDP. Au cours de la réaction catalysée par la pyruvate kinase, l'ADP et le phosphoénolpyruvate donnent de l'ATP et du pyruvate. Ce dernier réagit avec de la phénylhydrazine qui se transforme en phénylhydrazone, composé détectable à 450 nm. Ce dosage peut être effectue sur plaque ELISA.In the article by Kezdi et al, mentioned above, the enzymatic activity is measured by a coupled enzymatic system comprising the pyruvate kinase which uses the ADP formed by the NDP kinase from ATP and DTDP. During the reaction catalyzed by pyruvate kinase, ADP and phosphoenolpyruvate give ATP and pyruvate. The latter reacts with phenylhydrazine which transforms into phenylhydrazone, a compound detectable at 450 nm. This assay can be performed on an ELISA plate.
L'activité NDP kinase peut également être suivie en utilisant des nucléotides naturels ou des analogues de synthèse radioactifs et des techniques de séparation des produits de la réaction par chro atographie (PARKS & AGARWAL, 1973, in The enzymes (BOYER P.D. Ed) , 8, 307,Académie Press, NY) .NDP kinase activity can also be followed using natural nucleotides or radioactive synthetic analogs and techniques for separation of reaction products by chromatography (PARKS & AGARWAL, 1973, in The enzymes (BOYER PD Ed), 8 , 307, Academy Press, NY).
Selon un autre mode avantageux de réalisation de la méthode de détection de l'invention,l'éventuel accroissement de la production de NDP kinases est détecté en mesurant la quantité de NDP kinases dans l'échantillon biologique étudié.According to another advantageous embodiment of the detection method of the invention, the possible increase in the production of NDP kinases is detected by measuring the amount of NDP kinases in the biological sample studied.
Par exemple, on mesure la quantité de NDP kinases par marquage de 1'intermédiaire phosphorylé résultant de 1'interaction du nucléotide triphosphate substrat avec l'enzyme, le marquage de l'intermédiaire phosphorylé étant effectué à l'aide d'analogues tels les dérivés 7- thiophosphates de l'ATP ou du GTP qui présentent l'avantage de ne pouvoir être hydrolyses par les phosphatases.For example, the quantity of NDP kinases is measured by labeling the phosphorylated intermediate resulting from the interaction of the nucleotide triphosphate substrate with the enzyme, the labeling of the phosphorylated intermediate being carried out using analogues such as derivatives. 7 - thiophosphates of ATP or GTP which present the advantage of not being able to be hydrolyzed by phosphatases.
Selon un autre mode de réalisation de l'invention, comme 1'augmentation d'activité observée correspond à un taux plus important d'enzyme, on mesure la quantité de NDP kinases présentes à l'aide d'anticorps anti-NDP kinases polyclonaux ou monoclonaux, et on met en évidence les éventuels complexes immunologiques formés entre les NDP kinases et les anticorps sus-mentionnés. Cette détection peut être effectuée sur coupe histologique ou après électrophorèse de l'échantillon et transfert de WESTERN, ou par tout autre moyen approprié.According to another embodiment of the invention, since the increase in activity observed corresponds to a higher level of enzyme, the amount of NDP kinases present is measured using polyclonal anti-NDP kinase antibodies or monoclonal, and the possible immunological complexes formed between the NDP kinases and the abovementioned antibodies are highlighted. This detection can be carried out on a histological section or after electrophoresis of the sample and transfer of WESTERN, or by any other appropriate means.
Les anticorps anti-NDP kinases sus-mentionnés sont choisis parmi les anticorps susceptibles de reconnaître toute NDP kinase ,et plus particulièrement parmi ceux dirigés contre toute ou partie des sous-unités A et/ou B des NDP kinases d'érythrocytes humains (Presecan, E et al. (1989) FEBS lett.250, 629-632), ou ceux dirigés contre toute ou partie des protéines décrites par Rosengard, A.M. et al. dans NATURE 342, 177-180 (1989) et dont il a été montré qu'elles correspondraient à des NDP kinases ( allet et al., J. Nat. Cancer Inst. (1990) 82, 1199-1202) , ou encore ceux dirigés contre toute ou partie de la NDP kinase représentée sur la figure 1, et qui est décrite ci-dessous.The anti-NDP kinase antibodies mentioned above are chosen from the antibodies capable of recognizing any NDP kinase, and more particularly from those directed against all or part of the A and / or B subunits of the NDP kinases of human erythrocytes (Presecan, E et al. (1989) FEBS lett. 250, 629-632), or those directed against all or part of the proteins described by Rosengard, AM et al. in NATURE 342, 177-180 (1989) and which it has been shown to correspond to NDP kinases (allet et al., J. Nat. Cancer Inst. (1990) 82, 1199-1202), or those raised against all or part of the NDP kinase shown in Figure 1, and which is described below.
Les systèmes révélateurs les plus usuels comprennent des marqueurs chimiques, physiques et/ou enzymatiques et/ou chimioluminescents détectables lorsque la réaction du type antigène-anticorps s'est produite. On citera les systèmes colori étriques couplés à des enzymes, les systèmes fluorescents ou encore radiomarqués.The most common developing systems include chemical, physical and / or enzymatic and / or chemiluminescent markers detectable when the antigen-antibody type reaction has occurred. Mention will be made of colorimetric systems coupled with enzymes, fluorescent or even radiolabelled systems.
A ce titre la présente invention concerne les anticorps anti-NDP kinases polyclonaux ou monoclonaux susceptibles de reconnaître toute ou partie de la protéine à activité NDP kinase (et de se lier immunologiquement avec cette protéine) telle qu'obtenue par clonage du gène codant pour la NDP kinase de Dictyostelium discoideum et purification de la protéine synthétisée.As such, the present invention relates to polyclonal or monoclonal anti-NDP kinase antibodies capable of recognizing all or part of the protein with NDP kinase activity (and to bind immunologically with this protein) as obtained by cloning the gene coding for NDP kinase from Dictyostelium discoideum and purification of the synthesized protein.
Cette protéine constitue 1•un des objets de 1'invention en tant que produit nouveau.This protein constitutes one of the objects of the invention as a new product.
Elle est caractérisée par l'enchaînement de 155 acides aminés représenté sur la figure 1. Son poids moléculaire théorique Mr est de 16700 ± 10 %.It is characterized by the sequence of 155 amino acids shown in Figure 1. Its theoretical molecular weight Mr is 16700 ± 10%.
La protéine à activité NDP kinase de 1'invention est encore caractérisée en ce qu'elle est fortement basique avec un pi calculé de 9,15, en ce qu'elle contient un fragment hydrophobe (résidus 64 à 84), et en ce qu'elle est particulièrement stable. Ses propriétés basiques permettent de la purifier aisément. Cette protéine constitue donc une source industrielle de NDP kinase présentant un grand intérêt par rapport aux NDP kinases obtenues actuellement à partir d'extraits de tissus ou d'organes et dont la purification nécessite la mise en oeuvre de techniques coûteuses.The protein with NDP kinase activity of the invention is further characterized in that it is strongly basic with a calculated pi of 9.15, in that it contains a hydrophobic fragment (residues 64 to 84), and in that 'It is particularly stable. Its basic properties allow it to be easily purified. This protein therefore constitutes an industrial source of NDP kinase of great interest compared to NDP kinases currently obtained from extracts of tissues or organs and the purification of which requires the implementation of expensive techniques.
Il va de soi que toute séquence peptidique issue de la modification, par substitution et/ou par addition et/ou suppression d'un ou plusieurs acides aminés d'une protéine à activité NDP kinase , et plus particulièrement de la séquence peptidique représentée sur la figure 1 , ou d'une sous-séquence peptidique issue de cette dernière, entre le cadre de la présente invention, dès lors que cette modification n'altère pas les propriétés enzymatiques du type NDP kinase dudit polypeptide.It goes without saying that any peptide sequence resulting from the modification, by substitution and / or by addition and / or deletion of one or more amino acids of a protein with NDP kinase activity, and more particularly of the peptide sequence represented on the Figure 1, or a peptide subsequence from the latter, between the scope of the present invention, since this modification does not alter the enzymatic properties of the NDP kinase type of said polypeptide.
L'invention vise également une séquence de nucléotides constituée par au moins une partie d'une séquence codant pour une protéine à activité NDP kinase telle qu'exprimée par D.discoideum.The invention also relates to a nucleotide sequence consisting of at least part of a sequence coding for a protein with NDP kinase activity as expressed by D. discoideum.
Cette séquence est capable de s'hybrider avec des gènes codant pour une protéine à activité NDP kinase.This sequence is capable of hybridizing with genes coding for a protein with NDP kinase activity.
Le pourcentage d'homologie en ce qui concerne les séquences qui s'hybrident est de 70 à 90 %.The percentage of homology with regard to the sequences which hybridize is from 70 to 90%.
Les conditions pour les hybridations évoquées en rapport avec les nouvelles séquences de nucléotides sont des conditions de stringence relativement faibles. On utilise ainsi par exemple 30 % de formamide, 6xSSC (citrate de sodium, NaCl) , 0,12 M de pyrophosphate de sodium, 0,5 % de SDS et 250 μg d'ADN de sperme de hareng à 30βC. Les lavages sont effectués dans le même milieu à 37°C et suivant l'importance du bruit de fond, en diminuant progressivement la concentration de SSC et en augmentant la température de lavage de 5*C en 5*C.The conditions for the hybridizations mentioned in connection with the new nucleotide sequences are relatively low stringency conditions. Thus, for example, 30% of formamide, 6xSSC (sodium citrate, NaCl), 0.12 M of sodium pyrophosphate, 0.5% of SDS and 250 μg of herring sperm DNA are used at 30 β C. washes are performed in the same medium at 37 ° C and according to the importance of the background noise, by gradually decreasing the concentration of SSC and increasing the wash temperature by 5 ° C 5 ° C.
L'invention concerne également une séquence recombinante comprenant l'une des séquences définies ci- dessus, le cas échéant associée à un promoteur capable de contrôler la transcription de la séquence d'ADN codant pour des séquences de terminaison de la transcription et des signaux de traduction et de sécrétion.The invention also relates to a recombinant sequence comprising one of the sequences defined above, optionally associated with a promoter capable of controlling the transcription of the DNA sequence coding for transcription termination sequences and translation and secretion.
Selon encore un autre aspect, la séquence de nucléotides de l'invention est capable de s'hybrider avec une sonde formée à partir de la séquence présentant 1*enchaînement de nucléotides représenté sur la figure 1 ou l'enchaînement des nucléotides complémentaires de cette séquence.According to yet another aspect, the nucleotide sequence of the invention is capable of hybridizing with a probe formed from the sequence having the sequence of nucleotides shown in FIG. 1 or the sequence of nucleotides complementary to this sequence .
Les séquences de 1'invention sont des séquences d'ADN ou d'ARN.The sequences of the invention are DNA or RNA sequences.
Toute séquence de nucléotides hybridable avec celle de l'enchaînement de la figure 1, telle qu'obtenue par transcription enzymatique inverse de l'ARN correspondant, ou encore par synthèse chimique, fait partie de l'invention.Any nucleotide sequence hybridizable with that of the sequence of FIG. 1, as obtained by reverse enzymatic transcription of the corresponding RNA, or also by chemical synthesis, is part of the invention.
Conformément à l'invention, le clonage de la séquence de nucléotides codant pour une protéine à activité NDP kinase comprend l'isolement des clones d'une banque d'expression construite dans un vecteur, du type de ceux exprimant une protéine interagissant spécifiquement avec un composé tel que [5S] GTP7S, donneur de triphosphate.According to the invention, the cloning of the nucleotide sequence coding for a protein with NDP kinase activity comprises the isolation of clones of an expression library constructed in a vector, of the type of those expressing a protein interacting specifically with a compound such as [ 5 S] GTP 7 S, triphosphate donor.
Pour l'expression de la protéine dans un hôte, on construit un vecteur en insérant un fragment nucléotidique renfermant la séquence codante.For the expression of the protein in a host, a vector is constructed by inserting a nucleotide fragment containing the coding sequence.
Selon un mode de réalisation avantageux, on construit par exemple le plasmide pNDK pour exprimer la protéine à activité NDP kinase dans une bactérie telle que E.coli. A cet effet, on insère le fragment de restriction HaelII - EçoRI du clone isolé, ce fragment ayant été avantageusement traité au préalable pour obtenir des extrémités franches. L'insertion est réalisée dans le site HincII du plasmide pTZ18R. Le plasmide recombinant résultant est introduit, selon les techniques habituelles, dans la bactérie aux fins d'expression de l'enzyme sous le contrôle du promoteur lac Z.According to an advantageous embodiment, the plasmid pNDK is constructed, for example, to express the protein with NDP kinase activity in a bacterium such as E. coli. To this end, the HaelII - EçoRI restriction fragment of the isolated clone is inserted, this fragment having been advantageously treated beforehand to obtain blunt ends. The insertion is carried out in the HincII site of the plasmid pTZ18R. The resulting recombinant plasmid is introduced, according to usual techniques, into the bacterium for the purpose of expression of the enzyme under the control of the lac Z promoter.
Un agent inducteur tel que l'IPTG est avantageusement ajouté pour augmenter le taux d'expression. Plusieurs mg par litre de culture peuvent être ainsi obtenus.An inducing agent such as IPTG is advantageously added to increase the level of expression. Several mg per liter of culture can thus be obtained.
L'enzyme récupérée est soumise à au moins deux étapes de purification sur une colonne de chromatographie.The enzyme recovered is subjected to at least two purification steps on a chromatography column.
Une préparation homogène d'enzyme est obtenue en mettant en contact un surnageant de centrifugation d'une suspension de bactéries préalablement cassées par sonication, obtenue à l'issue du procédé ci-dessus, avec un dérivé de dextrane tel que le DEAE-Sephacel®, (à pH 8.5, la protéine à activité NDP kinase n'est pas retenue dans la résine de DEAE et est éluée dans le volume mort) , l'éluat recueilli est ensuite chro atographié sur une colonne de résine telle que Affigel-Blue®, et élue avec 0,7M NaCl.A homogeneous enzyme preparation is obtained by contacting a centrifugation supernatant of a suspension of bacteria previously broken by sonication, obtained at the end of the above process, with a dextran derivative such as DEAE-Sephacel ®, (at pH 8.5, the protein NDP kinase activity is not retained in the DEAE resin and is eluted in the void volume), the eluate is then collected chro atographié on a resin column such as Affigel-Blue ® , and eluted with 0.7M NaCl.
Les applications des produits selon l'invention comprennent également l'élaboration, à partir de fragments de l'enchaînement de nucléotides de la figure 1, de sondes d'ARN, d'ARNm ou d'ADN, pour la détection de séquences similaires dans d'autres organismes ou tissus.The applications of the products according to the invention also include the development, from fragments of the nucleotide sequence of FIG. 1, of RNA, mRNA or DNA probes, for the detection of similar sequences in other organisms or tissues.
Cette élaboration comprend, notamment, la dénaturation des séquences double brin pour obtenir une séquence monobrin utilisable en tant que sonde.This elaboration includes, in particular, the denaturation of the double-stranded sequences to obtain a single-stranded sequence usable as a probe.
L'invention vise donc des sondes de détection caractérisées en ce qu'elles comprennent au moins une partie d'une séquence de nucléotides définie ci-dessus, d'ARN ou d'ADN, plus spécialement un fragment d'une séquence codant pour une protéine à activité NDP kinase.The invention therefore relates to detection probes characterized in that they comprise at least part of a nucleotide sequence defined above, of RNA or DNA, more especially a fragment of a sequence coding for a protein with NDP kinase activity.
Le fragment utilisé comme sonde comporte un nombre de nucléotides suffisant pour obtenir la spécificité reguise et la formation d'un hybride stable.The fragment used as probe contains a sufficient number of nucleotides to obtain the specificity and to form a stable hybrid.
Des sondes appropriées sont avantageusement marquées par un élément radioactif (sondes chaudes) ou tout autre groupe non radioactif (sondes froides) permettant la reconnaissance de la sonde à l'état hybride avec la préparation renfermant l'ADN à étudier.Appropriate probes are advantageously marked with a radioactive element (hot probes) or any other non-radioactive group (cold probes) allowing recognition of the probe in a hybrid state with the preparation containing the DNA to be studied.
Selon encore un autre mode de réalisation de l'invention, on utilise des sondes d'ADNc spécifiques des NDP kinases du type de celles décrites ci-dessus, notamment des sondes capables de s'hybrider avec les séquences correspondantes des ARNm codant pour les NDP kinases, une augmentation du taux des ARNm pouvant être reliée à une augmentation de la quantité de protéines NDP kinases reflétant l'état tumoral de l'échantillon.According to yet another embodiment of the invention, cDNA probes specific for NDP kinases of the type described above are used, in particular probes capable of hybridizing with the corresponding sequences of the mRNAs coding for NDP kinases, an increase in the level of mRNA may be linked to an increase in the amount of NDP kinase proteins reflecting the tumor state of the sample.
Avantageusement, l'activité ou la quantité de NDP kinases est mesurée sur un échantillon biologique provenant d'un patient.Advantageously, the activity or the quantity of NDP kinases is measured on a biological sample originating from a patient.
Pour la mise en évidence d'un état tumoral, on effectue plus spécialement une comparaison d'activité entre un échantillon biologique contenant des cellules hautement prolifératives et un échantillon contenant des cellules correspondantes normales.For the detection of a tumor state, an activity comparison is more particularly carried out between a biological sample containing highly proliferative cells and a sample containing normal corresponding cells.
Par échantillon biologique, on entend tout prélèvement effectué sur le patient qu'il s'agisse d'une tumeur solide, d'un fluide biologique tel que le sang, le sérum, le liquide céphalo-rachidien, le liquide pleural, l'urine, les crachats, des prélèvements obtenus par tubage, aspiration bronchique, ou ponction, ou encore des cellules circulantes.By biological sample is meant any sample taken from the patient, whether it is a solid tumor or a biological fluid such as blood, serum, cerebrospinal fluid, pleural fluid, urine. , sputum, samples obtained by tubing, bronchial aspiration, or puncture, or circulating cells.
Pour effectuer la mesure, l'échantillon biologique est utilisé sous forme entière, broyée, en coupe, fractionnée, en particulier sous forme de fractions sub¬ cellulaires ou, le cas échéant, sous forme de culture cellulaire.To carry out the measurement, the biological sample is used in whole, crushed, sectioned, fractional form, in particular in the form of sub¬ cell fractions or, where appropriate, in the form of cell culture.
Les réactifs nécessaires à la mise en évidence de 1*activité ou de la quantité de NDP kinases peuvent être dans un milieu liquide ou incorporés dans un matériau support par exemple des gels ou des filtres-support.The reagents necessary for demonstrating the activity or the amount of NDP kinases can be in a liquid medium or incorporated into a support material, for example gels or support filters.
L'invention vise également des kits pour la détection in vitro de tumeurs malignes.The invention also relates to kits for the in vitro detection of malignant tumors.
Ces kits sont caractérisés en ce qu'ils renferment les réactifs nécessaires tels que définis ci-dessus pour la mise en évidence de l'éventuel accroissement de la production de NDP kinases lorsque ces réactifs sont mis en contact avec un échantillon biologique sous forme appropriée. Les kits de détection de 1'invention présentent l'avantage de pouvoir être utilisés directement sur des échantillons cliniques et permettent d'obtenir des résultats dans un temps très rapide.These kits are characterized in that they contain the necessary reagents as defined above for demonstrating the possible increase in the production of NDP kinases when these reagents are brought into contact with a biological sample in appropriate form. The detection kits of the invention have the advantage of being able to be used directly on clinical samples and allow results to be obtained in a very rapid time.
En fonction des différentes méthodes de détection de l'invention décrites ci-dessus, l'invention concerne des kits de détection caractérisés en ce qu'ils renferment :Depending on the different detection methods of the invention described above, the invention relates to detection kits characterized in that they contain:
- des nucleosides diphosphates et triphosphates naturels ou des analogues de synthèse de ces nucléotides, le cas échéant radioactivement marqués, ou sous forme de dérivés 7-thiophosphates de l'ATP ou du GTP,- natural nucleoside diphosphates and triphosphates or analogs of synthesis of these nucleotides, optionally radioactive labeled, or in the form of 7- thiophosphate derivatives of ATP or of GTP,
- un milieu réactionnel approprié pour la réaction enzymatique entre les nucléotides et les NDP kinases, des systèmes enzymatiques couplés permettant la synthèse de produits détectables par colorimétrie et/ou spectrophotométrie, à des dérivés 7-thiophosphates de l'ATP ou de GTP permettant le marquage de l'intermédiaire phosphorylé, ou des kits renfermant : des anticorps anti-NDP kinases polyclonaux ou monoclonaux , le cas échéant marqués , notamment de manière radioactive ou enzymatique,- a suitable reaction medium for the enzymatic reaction between nucleotides and NDP kinases, coupled enzymatic systems allowing the synthesis of products detectable by colorimetry and / or spectrophotometry, to 7- thiophosphates derivatives of ATP or of GTP allowing labeling of the phosphorylated intermediate, or of the kits containing: polyclonal or monoclonal anti-NDP kinase antibodies, where appropriate labeled, in particular in a radioactive or enzymatic manner,
- un milieu approprié pour la réaction immunologique entre les susdits anticorps et les NDP kinases,- a medium suitable for the immunological reaction between the above-mentioned antibodies and the NDP kinases,
- des réactifs appropriés pour la détection des complexes immunologiques formés entre les susdits anticorps et les NDP kinases, ou encore des kits comprenant :- reagents suitable for the detection of the immunological complexes formed between the abovementioned antibodies and the NDP kinases, or also kits comprising:
- des sondes d'ADNc spécifiques des NDP kinases,le cas échéant marquées , notamment de manière radioactive ou enzymatique,- cDNA probes specific for NDP kinases, where appropriate labeled, in particular in a radioactive or enzymatic manner,
- un milieu approprié pour la réaction d'hybridation entre les susdits anticorps et les ARNm codant pour les NDP-kinases, - des réactifs appropriés pour la détection des sondes sus-mentionnées hybridées aux ARNm codant pour les NDP kinases.a medium suitable for the hybridization reaction between the above-mentioned antibodies and the mRNAs coding for the NDP-kinases, - reagents suitable for the detection of the above-mentioned probes hybridized to mRNAs coding for NDP kinases.
L'invention concerne également des compositions pharmaceutiques comprenant toute ou partie d'une (ou plusieurs) protéine à activité NDP kinase, notamment celle obtenue par clonage du gène codant pour la NDP kinase de Dictyos e1ium discoideum et purification de la protéine synthétisée, ou encore celle correspondant aux sous-unités A et/ou B des érythrocytes humains (cf.Presecan et al., et Rosengard et al. sus-mentionnés) en association avec un véhicule physiologiquement acceptable.The invention also relates to pharmaceutical compositions comprising all or part of one (or more) protein with NDP kinase activity, in particular that obtained by cloning the gene coding for NDP kinase from Dictyos e1ium discoideum and purification of the synthesized protein, or alternatively that corresponding to subunits A and / or B of human erythrocytes (cf. Presecan et al., and Rosengard et al. mentioned above) in association with a physiologically acceptable vehicle.
L'invention concerne plus particulièrement des compositions pharmaceutiques comprenant toute ou partie de la séquence d'acides aminés représentée sur la figure 1 en association avec un véhicule physiologiquement acceptable.The invention relates more particularly to pharmaceutical compositions comprising all or part of the amino acid sequence shown in FIG. 1 in association with a physiologically acceptable vehicle.
L'invention a également pour objet des compositions pharmaceutiques comprenant des anticorps anti-NDP kinases polyclonaux ou monoclonaux tels que désignés ci-dessus, en association avec un véhicule physiologiquement acceptable.A subject of the invention is also pharmaceutical compositions comprising anti-NDP kinase polyclonal or monoclonal antibodies as designated above, in association with a physiologically acceptable vehicle.
L'invention vise également des compositions pharmaceutiques comprenant des séquences nucléotidiques telles que définies ci-dessus , et plus particulièrement des séquences nucléotidiques antisens complémentaires de toute ou partie de la séquence nucléotidique codant pour une protéine à activité NDP kinase, notamment de la séquence nucléotidique représentée sur la figure 1, en association avec un véhicule physiologiquement acceptable.The invention also relates to pharmaceutical compositions comprising nucleotide sequences as defined above, and more particularly antisense nucleotide sequences complementary to all or part of the nucleotide sequence coding for a protein with NDP kinase activity, in particular of the nucleotide sequence shown in Figure 1, in association with a physiologically acceptable vehicle.
Les compositions pharmaceutiques sus-mentionnées de l'invention sont plus particulièrement destinées au traitement des tumeurs et notamment à la prévention de l'évolution d'une tumeur bénigne vers une tumeur maligne. Ces compositions pharmaceutiques présentent l'avantage d'inhiber l'activité NDP kinase.The above-mentioned pharmaceutical compositions of the invention are more particularly intended for treatment of tumors and in particular the prevention of the progression from a benign tumor to a malignant tumor. These pharmaceutical compositions have the advantage of inhibiting NDP kinase activity.
L'invention concerne également toute séquence nucléique comprise dans l'une des séquences nucléiques définies ci-dessus, ou susceptible de s'hybrider avec l'une de ces séquences dans les conditions d'hybridation définies ci-dessus, et étant utilisable en tant qu'amorce nucléique pour l'amplification génique d'une des séquences nucléiques de l'invention.The invention also relates to any nucleic acid sequence included in one of the nucleic acid sequences defined above, or capable of hybridizing with one of these sequences under the hybridization conditions defined above, and being usable as as a nucleic acid primer for the gene amplification of one of the nucleic acid sequences of the invention.
Avantageusement les amorces nucléiques de l'invention sont constituées d'une succession d'environ 10 à 30 nucléotides.Advantageously, the nucleic acid primers of the invention consist of a succession of approximately 10 to 30 nucleotides.
Les techniques d'amplification génique sont d'un appoint considérable pour la mise au point de méthodes de diagnostic in vitro particulièrement sensibles. Parmi ces techniques d'amplification génique, on peut citer la technique PCR (Poly erase Chain Reaction) telle que décrite dans les demandes de brevet européen n' 86/302.298.4 du 27/03/1986 et nβ 87/300.203.4 du 09/01/1987, ou encore la technique dite "Q/3replicase" décrite dans Biotechnology, vol.6, page 1197 (octobre 1988) et celle procédant à l'aide d'une ARN polymerase (T7RNA polymerase) décrite danés la demande de brevet international n" WO89/01050. Ces techniques permettent d'améliorer la sensibilité de détection des acides nucléiques des virus ou des bactéries, et nécessitent l'utilisation d'amorces de synthèse spécifiques.Gene amplification techniques are a considerable adjunct to the development of particularly sensitive in vitro diagnostic methods. Among these gene amplification techniques, mention may be made of the PCR (Poly erase Chain Reaction) technique as described in European patent applications No. 86 / 302.298.4 of 03/27/1986 and No. β 87 / 300.203.4 01/09/1987, or the technique called "Q / 3replicase" described in Biotechnology, vol.6, page 1197 (October 1988) and that using a RNA polymerase (T7RNA polymerase) described in the international patent application No. WO89 / 01050. These techniques make it possible to improve the detection sensitivity of nucleic acids of viruses or bacteria, and require the use of specific synthetic primers.
Les anticorps polyclonaux de 1'invention sont obtenus par immunisation d'un animal avec les polypeptides de l'invention, suivie de la récupération des anticorps formés. Les anticorps monoclonaux de l'invention sont produits par tout hybridome susceptible d'être formé, par des méthodes classiques, à partir des cellules εpléniques d'un animal, notamment de souris ou de rat, immunisés contre l'un des polypeptides purifiés de l'invention, d'une part et des cellules d'une lignée de cellules d'un myélome approprié d'autre part, et d'être sélectionné, par sa capacité à produire des anticorps monoclonaux reconnaissant le polypeptide initialement mis en oeuvre pour l'immunisation des animaux.The polyclonal antibodies of the invention are obtained by immunization of an animal with the polypeptides of the invention, followed by the recovery of the antibodies formed. The monoclonal antibodies of the invention are produced by any hybridoma capable of being formed, by conventional methods, from the εplenic cells of an animal, in particular mouse or rat, immunized against one of the purified polypeptides of l invention, on the one hand and cells of a cell line of a suitable myeloma on the other hand, and to be selected, by its capacity to produce monoclonal antibodies recognizing the polypeptide initially used for the animal immunization.
Un procédé de préparation particulièrement avantageux des séquences nucléiques de 1'invention comprend les étapes suivantes :A particularly advantageous process for preparing the nucleic acid sequences of the invention comprises the following steps:
- la synthèse d'ADN en utilisant la méthode automatisée des 3-cyanethyl phosphora idite décrite dans Bioorganic Chemistry 4; 274-325 (1986),- DNA synthesis using the automated 3-cyanethyl phosphora idite method described in Bioorganic Chemistry 4; 274-325 (1986),
- le clonage des acides nucléiques ainsi obtenus dans un vecteur approprié et la récupération de l'acide nucléique par hybridation avec une sonde appropriée choisie parmi celles décrites ci-dessus.- Cloning of the nucleic acids thus obtained into an appropriate vector and recovery of the nucleic acid by hybridization with an appropriate probe chosen from those described above.
Un autre procédé de préparation des séquences nucléotidiques de l'invention comprend les étapes suivantes :Another method for preparing the nucleotide sequences of the invention comprises the following steps:
- l'assemblage d'oligonucléotides synthétisés chimiquement, pourvus à leurs extrémités de sites de restriction différents, dont les séquences sont compatibles avec l'enchaînement en acides aminés du polypeptide naturel selon le principe décrit dans Proc. Natl. Acad. Sci. USA, 80; 7461-7465, (1983) ,- The assembly of chemically synthesized oligonucleotides, provided at their ends with different restriction sites, whose sequences are compatible with the amino acid sequence of the natural polypeptide according to the principle described in Proc. Natl. Acad. Sci. USA, 80; 7461-7465, (1983),
- le clonage des acides nucléiques ainsi obtenus dans un vecteur approprié et la récupération de l'acide nucléique recherché par hybridation avec une sonde appropriée choisie parmi celles décrites ci-dessus. L'invention a également pour objet tout acide nucléique recombinant contenant au moins une séquence nucléique de l'invention, insérée dans un acide nucléique hétérologue vis-à-vis de ladite séquence nucléique.- The cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the nucleic acid sought by hybridization with an appropriate probe chosen from those described above. The subject of the invention is also any recombinant nucleic acid containing at least one nucleic sequence of the invention, inserted into a nucleic acid heterologous with respect to said nucleic sequence.
L'invention concerne plus particulièrement un acide nucléique recombinant tel que défini ci-dessus, dans lequel la séquence nucléique de 1•invention est précédée d'un promoteur (notamment un promoteur inductible) sous le contrôle duquel la transcription de ladite séquence est susceptible d'être effectuée et, le cas échéant, suivie d'une séquence codant pour des signaux de terminaison de la transcription.The invention relates more particularly to a recombinant nucleic acid as defined above, in which the nucleic sequence of the invention is preceded by a promoter (in particular an inducible promoter) under the control of which the transcription of said sequence is capable of be performed and, where appropriate, followed by a sequence encoding transcription termination signals.
L'invention concerne tout vecteur recombinant, utilisé en particulier pour le clonage d'une séquence nucléique de l'invention, et/ou l'expression du polypeptide codé par cette séquence, et caractérisé en ce qu'il contient un acide nucléique recombinant, tel que défini ci-dessus, en l'un de ses sites non essentiel pour sa réplication.The invention relates to any recombinant vector, used in particular for the cloning of a nucleic sequence of the invention, and / or the expression of the polypeptide encoded by this sequence, and characterized in that it contains a recombinant nucleic acid, as defined above, at one of its sites not essential for its replication.
A titre d'exemple de vecteur sus- entionné, on citera les plas ides, les cosmides, ou les phages.By way of example of the abovementioned vector, mention will be made of plasids, cosmids, or phages.
L'invention a également pour objet un procédé de préparation d'un polypeptide de l'invention, par transformation d'un hôte cellulaire à l'aide d'un vecteur recombinant de type sus-indiqué, suivie de la mise en culture de l'hôte cellulaire ainsi transformé, et de la récupération du polypeptide dans le milieu de culture.The subject of the invention is also a process for the preparation of a polypeptide of the invention, by transformation of a cellular host using a recombinant vector of the above-mentioned type, followed by the cultivation of the cell host thus transformed, and recovery of the polypeptide from the culture medium.
Ainsi, l'invention concerne tout hôte cellulaire transformé par un vecteur recombinant tel que défini ci- dessus, et comprenant les éléments de régulation permettant l'expression de la séquence nucléique codant pour un polypeptide selon l'invention.Thus, the invention relates to any cellular host transformed by a recombinant vector as defined above, and comprising the regulatory elements allowing the expression of the nucleic sequence coding for a polypeptide according to the invention.
Les peptides selon 1'invention peuvent être préparés par les techniques classiques, dans le domaine de la synthèse des peptides. Cette synthèse peut être réalisée en solution homogène ou en phase solide.The peptides according to the invention can be prepared by conventional techniques, in the field of peptide synthesis. This synthesis can be carried out in homogeneous solution or in solid phase.
Par exemple, on aura recours à la technique de synthèse en solution homogène décrite par HOUBENWEYL dans l'ouvrage intitulé "Méthode der Organischen Che ie" (Méthode de la Chimie Organique) édité par E. Wunsch, vol. 15-1 et II., THIEME, Stuttgart 1974.For example, we will use the synthesis technique in homogeneous solution described by HOUBENWEYL in the work entitled "Method der Organischen Che ie" (Method of Organic Chemistry) edited by E. Wunsch, vol. 15-1 and II., THIEME, Stuttgart 1974.
Selon une autre technique préférée de l'invention, on a recours à la synthèse en phase solide décrite par R.D. MERRIFIELD dans l'article intitulé "Solid phase peptide synthesis" (J. A . Che . Soc. , 4_5, 2149-2154) .According to another preferred technique of the invention, use is made of the solid phase synthesis described by RD MERRIFIELD in the article entitled "Solid phase peptide synthesis" (J. A. Che. Soc., 4_5, 2149-2154) .
On rapporte dans les exemples ci-après donnés à titre illustratif une méthode de détection de l'activité NDP kinase mesurée sur des extraits de tissus de différents patients. Les résultats obtenus sont rapportés sur la figure 2 et le tableau 1 qui concernent les variations de l'activité de la NDPK en U/mg de protéine selon le tissu étudié pour deux séries de patients. EXEMPLES Préparation des extraitsIn the examples given below by way of illustration, a method of detecting NDP kinase activity measured on tissue extracts from different patients is reported. The results obtained are reported in FIG. 2 and Table 1 which relate to the variations in the activity of NDPK in U / mg of protein according to the tissue studied for two series of patients. EXAMPLES Preparation of extracts
Les fragments de tissus obtenus congelés dans de l'azote liquide sont dégelés rapidement dans du tampon Tris-HCl 10 mM, pH 7,5 maintenu à 4'C, contenant de l'EDTA 1 mM, du glycérol 20% et des inhibiteurs de protéases (PMSF (fluorure de phényl méthyl sulfonyl) 1 mM, 1 μg/ml d'antipaïn et 15 μg/ml de benzamidine) à raison 5 ml environ de tampon par gramme de tissus. Les tissus sont coupés finement puis broyés au Polytron® (3 fois 20 secondes à puissance maximale) . Le broyât est ensuite centrifugé à 4"C, 10 min. à 20000 g dans une centrifugeuse réfrigérée. Le surnageant est utilisé immédiatement ou conservé par aliquots à -80°C. La concentration en protéines de celui-ci est mesurée par la méthode de BRADFORD (1976, Anal. Biochem., 72, 248). Dosage de l'activité NDP kinaseThe tissue fragments obtained frozen in liquid nitrogen are quickly thawed in 10 mM Tris-HCl buffer, pH 7.5 maintained at 4 ° C., containing 1 mM EDTA, 20% glycerol and inhibitors of proteases (PMSF (phenyl methyl sulfonyl fluoride) 1 mM, 1 μg / ml of antipain and 15 μg / ml of benzamidine) at a rate of approximately 5 ml of buffer per gram of tissue. The fabrics are finely cut and then ground with Polytron® (3 times 20 seconds at maximum power). The ground material is then centrifuged at 4 "C., 10 min. At 20,000 g in a refrigerated centrifuge. The supernatant is used immediately or stored in aliquots at -80 ° C. protein concentration thereof is measured by the method of BRADFORD (1976, Anal. Biochem., 72, 248). Determination of NDP kinase activity
L'activité enzymatique est mesurée par un système enzymatique couplé pyruvate kinase-lactate deshydrogenase (AGARWAL et al, 1978, Meth. Enzy ol. , 51, 376) qui relie la formation d'ADP par la (ou les) NDP kinase(s) à une consommation de NADH détectée spectrophotométriquement à 340 n . Les réactions sont les suivantes :The enzymatic activity is measured by an enzyme system coupled with pyruvate kinase-lactate dehydrogenase (AGARWAL et al, 1978, Meth. Enzy ol., 51, 376) which links the formation of ADP by the NDP kinase (s) ) to a consumption of NADH detected spectrophotometrically at 340 n. The reactions are as follows:
NDP kinaseNDP kinase
dTDP + ATP > dTTP + ADPdTDP + ATP> dTTP + ADP
PKPK
ADP + PEP > ATP + pyruvateADP + PEP> ATP + pyruvate
LDHLDH
Pyruvate + NADH —> lactate + NAD+ Pyruvate + NADH -> lactate + NAD +
(Abréviations: PEP: phosphoénolpyruvate; PK: pyruvate kinase; LDH: lactate deshydrogenase; dTDP: déoxythymidine diphosphate; dTTP: déoxythymidinetriphosphate)(Abbreviations: PEP: phosphoenolpyruvate; PK: pyruvate kinase; LDH: lactate dehydrogenase; dTDP: deoxythymidine diphosphate; dTTP: deoxythymidinetriphosphate)
La réaction a lieu à 30°C dans un volume total de 500 μl de Tris-HCl 50 mM, pH 7,4, contenant du phosphoé¬ nolpyruvate 0,5 mM, de l'ATP 0,5 mM, du MgCl2 6 mM, 100 μg/ml de NADH, du KC1 50 mM, du dTDP 0,2 mM, 1,5 U de pyruvate kinase et de lactate deshydrogenase et la quantité de surnageant d'extrait tisεulaire (1 à 10 μl) nécessaire à l'obtention d'une vitesse directement proportionnelle à la quantité d'extrait. Une .unité d'enzyme catalyse la formation d'une μmole d'ADP par minute.The reaction takes place at 30 ° C in a total volume of 500 μl of 50 mM Tris-HCl, pH 7.4, containing 0.5 mM phosphoéololpyruvate, 0.5 mM ATP, MgCl 2 6 mM, 100 μg / ml of NADH, 50 mM KC1, 0.2 mM dTDP, 1.5 U of pyruvate kinase and lactate dehydrogenase and the quantity of tisεular extract supernatant (1 to 10 μl) necessary for l '' obtaining a speed directly proportional to the amount of extract. A unit of enzyme catalyzes the formation of one μmole of ADP per minute.
En variante, ces mesures ont été réalisées à 30°C dans un milieu réactionnel de 0,5 ml constitué de 50mM tris HC1, pH 7,4, 50 mM KCl, 6 mM MgCl2, 1 mM phosphoénolpyruvate, 0,1 mg/ml NADH, 0,5 mM ATP, 0,1 mM dTDP, 2 unités de pyruvate kinase et 2,5 unités de lactate deshydrogenase. RésultatsAs a variant, these measurements were carried out at 30 ° C. in a 0.5 ml reaction medium consisting of 50 mM tris HCl, pH 7.4, 50 mM KCl, 6 mM MgCl 2 , 1 mM phosphoenolpyruvate, 0.1 mg / ml NADH, 0.5 mM ATP, 0.1 mM dTDP, 2 units of pyruvate kinase and 2.5 units of lactate dehydrogenase. Results
L'activité NDP kinase d'extraits de plusieurs tumeurs solides du sein a été comparée à celle de différents tissus normaux par la méthode pyruvate kinase-lactate deshydrogenase.The NDP kinase activity of extracts from several solid breast tumors was compared with that of different normal tissues by the pyruvate kinase-lactate dehydrogenase method.
On rapporte sur la figure 2 et le tableau 1 l'activité NDP kinase d'extraits de ganglions lymphatiques normaux (B7,H2), de muscles normaux (D11,A2) , de peau normale (A3) , de seins normaux (A5,B8,J6,K8) , de thyroïde normale (E12) , de tumeurs bénignes du sein (Gl,O5,N4,Q7,T10) , de tumeurs malignes du sein (A1,B6,F13,H3,J7,K9,V12,P6) ,de tumeurs malignes de ganglions lymphatiques (A4,U11), de tumeurs malignes du colon (L2,R8), et de tumeurs malignes du col de l'utérus (M13).The NDP kinase activity of extracts from normal lymph nodes (B7, H2), normal muscles (D11, A2), normal skin (A3), normal breasts (A5, B8, J6, K8), normal thyroid (E12), benign breast tumors (Gl, O5, N4, Q7, T10), malignant breast tumors (A1, B6, F13, H3, J7, K9, V12 , P6), malignant neoplasms of lymph nodes (A4, U11), malignant neoplasms of the colon (L2, R8), and malignant neoplasms of the cervix (M13).
L'examen de ces résultats montre que l'activité est augmentée de 5 à 10 fois par rapport à l'activité du tissu normal de référence.Examination of these results shows that the activity is increased by 5 to 10 times compared to the activity of the normal reference tissue.
L'augmentation de l'activité enzymatique NDP kinase correspond à une augmentation de la quantité de l'enzyme (et plus particulièrement de la sous-unité A de la NDP kinase d'érythrocytes humains), comme il a été évalué par transfert de WESTERN, en utilisant des anticorps spécifiques de NDP kinase d'érythrocytes humains. Ces résultats sont confirmés sur coupe de tissus par un marquage spécifique des cellules tumorales. L'activité NDP kinase a été mesurée dans des extraits de nombreux tissus normaux ou provenant de tumeurs malignes ou bénignes. Une augmentation remarquable et spécifique de la production de NDP kinase a été observée dans les tumeurs malignes (cf figure 2) . L'activité NDP kinase des tissus normaux varie entre 0,1 et 0,3 unité (U) par mg de protéine. L'activité NDP kinase est nettement supérieure dans les extraits de tumeurs malignes, tandis que les valeurs mesurées dans les extraits de tumeurs bénignes sont similaires à celles mesurées dans les tissus normaux. La plupart des valeurs trouvées dans les tumeurs malignes varient entre 0,5 à 0,9 U/mg de protéine. La plus forte activité a été mesurée dans une tumeur du sein (carcimone intracanalaire non infiltrant (P6)) et correspond à 1,93 U/mg de protéine. La plus faible valeur, 0,25 U/mg de protéine, a été trouvée dans 1'adénocarcimone (R8) du colon.The increase in the NDP kinase enzyme activity corresponds to an increase in the quantity of the enzyme (and more particularly of the A sub-unit of the NDP kinase of human erythrocytes), as has been evaluated by WESTERN transfer , using antibodies specific for NDP kinase from human erythrocytes. These results are confirmed on tissue section by specific labeling of tumor cells. NDP kinase activity has been measured in extracts from many normal tissues or from malignant or benign tumors. A remarkable and specific increase in the production of NDP kinase has been observed in malignant tumors (see Figure 2). The NDP kinase activity of normal tissues varies between 0.1 and 0.3 units (U) per mg of protein. The NDP kinase activity is clearly higher in the extracts of malignant tumors, while the values measured in the extracts of benign tumors are similar to those measured in normal tissues. Most of the values found in malignant tumors range from 0.5 to 0.9 U / mg protein. The highest activity was measured in a breast tumor (non-infiltrating intraductal carcimone (P6)) and corresponds to 1.93 U / mg of protein. The lowest value, 0.25 U / mg protein, was found in colon adenocarcimone (R8).
La mesure de l'activité NDP kinase constitue donc un critère d'évaluation de l'état tumoral évolutif potentiel d'un prélèvement tissulaire.The measurement of NDP kinase activity therefore constitutes a criterion for evaluating the potential progressive tumor state of a tissue sample.
L'invention fournit ainsi un test simple de dépistage d'états tumoraux potentiellement évolutifs.The invention thus provides a simple test for screening for potentially progressive tumor states.
Tableau 1 Table 1
Tissus Code Histologie Marquage NDP kinase Immunol. (U/mgprot.)Tissues Histology Code Marking NDP kinase Immunol. (U / mgprot.)
(a) (b) (a) (a)(a) (b) (a) (a)
cervix M3 c.indiff. +++ 0.58 gang.L. Ull mélan.métast. ++++ 0.80 cervix M3 vs. indiff. +++ 0.58 gang.L. Ull melan.metast. ++++ 0.80
(a) les abbreviations sont les suivantes :(a) the abbreviations are as follows:
- gang.L. : ganglion lymphatique- gang.L. : lymph node
- c. : carcinome- vs. : carcinoma
- métast. : métastatique- metast. : metastatic
- médull. : médullaire- medulla. : medullary
- canal.inf. : canalaire infiltrant- canal.inf. : infiltrating duct
- canal.n.inf. : canalaire non infiltrant- canal.n.inf. : non-infiltrating duct
- mélan. : mélanome- melan. : melanoma
- ND :non déterminé- ND: not determined
- i munol : immunologique- i munol: immunological
(b) les différents cas sont désignés par des codes (une même lettre capitale désigne le même patient) (b) the different cases are designated by codes (the same capital letter designates the same patient)

Claims

REVENDICATIONS
1. Méthode de détection in vitro de tumeurs malignes, réalisée à partir d'un échantillon biologique susceptible de présenter une telle tumeur , notamment un prélèvement de tissu effectué chez un patient , caractérisée en ce que l'on mesure l'éventuel accroissement de la production de NDP kinases dans cet échantillon biologique, par rapport aux NDP kinases normalement produites dans un échantillon biologique sain de référence, de même nature que l'échantillon biologique étudié sus-mentionné.1. Method for in vitro detection of malignant tumors, carried out from a biological sample capable of presenting such a tumor, in particular a tissue sample taken from a patient, characterized in that the possible increase in the production of NDP kinases in this biological sample, compared to the NDP kinases normally produced in a healthy reference biological sample, of the same nature as the biological sample studied above.
2. Méthode selon la revendication 1, caractérisée en ce que 1'éventuel accroissement de la production de NDP kinases est détecté en mesurant 1'activité NDP kinase dans l'échantillon biologique étudié.2. Method according to claim 1, characterized in that the possible increase in the production of NDP kinases is detected by measuring the NDP kinase activity in the biological sample studied.
3. Méthode selon la revendication 2, caractérisée en ce qu'elle est réalisée en mettant en présence l'échantillon biologique étudié avec des nucleosides diphosphates et triphosphates naturels ou des analogues de synthèse de ces nucléotides, et en mettant en évidence les éventuels produits de la réaction entre ces nucléotides, catalysée par les NDP kinases, par des systèmes enzymatiques couplés permettant la synthèse de produits détectables par colorimétrie et/ou spectrophotométrie.3. Method according to claim 2, characterized in that it is carried out by bringing together the biological sample studied with natural diphosphates and triphosphates nucleosides or analogs of synthesis of these nucleotides, and by highlighting the possible products of the reaction between these nucleotides, catalyzed by NDP kinases, by coupled enzymatic systems allowing the synthesis of products detectable by colorimetry and / or spectrophotometry.
4. Méthode selon la revendication 2, caractérisée en ce qu'elle est réalisée par mise en présence de l'échantillon biologique étudié avec des nucleosides diphosphates et triphosphates radioactivement marqués, naturels, ou des analogues de synthèse de ces nucléotides et en ce que l'on sépare les éventuels produits de la réaction, catalysée par les NDP kinases, par chromatographie. 4. Method according to claim 2, characterized in that it is carried out by bringing together the biological sample studied with radioactive labeled, natural nucleoside diphosphates and triphosphates, or analogs of synthesis of these nucleotides and in that l the possible products of the reaction, catalyzed by NDP kinases, are separated by chromatography.
5. Méthode selon la revendication 1, caractérisée en ce que l'éventuel accroissement de la production de NDP kinases est détecté en mesurant la quantité de NDP kinases dans l'échantillon biologique étudié.5. Method according to claim 1, characterized in that the possible increase in the production of NDP kinases is detected by measuring the amount of NDP kinases in the biological sample studied.
6. Méthode selon la revendication 5, caractérisée en ce qu'elle est réalisée par mise en présence de l'échantillon biologique étudié avec des nucleosides diphosphates et triphosphates naturels ou des analogues de synthèse de ces nucléotides, et par marquage de 1'intermédiaire phosphorylé résultant de 1'interaction du nucléoside triphosphate substrat avec l'enzyme, le marquage de 1'intermédiaire phosphorylé étant effectué à l'aide d'analogues tels les dérivés 7-thiophosphates de l'ATP ou du GTP.6. Method according to claim 5, characterized in that it is carried out by bringing the biological sample studied with natural nucleoside diphosphates and triphosphates or analogs of synthesis of these nucleotides into contact, and by labeling the phosphorylated intermediate resulting from the interaction of the nucleoside triphosphate substrate with the enzyme, the labeling of the phosphorylated intermediate being carried out using analogues such as the 7- thiophosphate derivatives of ATP or of GTP.
7. Méthode selon la revendication 5, caractérisée en ce qu'elle est réalisée par mise en présence de l'échantillon biologique étudié avec des anticorps anti- NDP kinases polyclonaux ou monoclonaux, et en ce que l'on met en évidence les éventuels complexes immunologiques formés entre les NDP kinases et les anticorps sus¬ mentionnés.7. Method according to claim 5, characterized in that it is carried out by bringing the biological sample studied with polyclonal or monoclonal anti-NDP kinase antibodies into contact, and in that any complexes are highlighted. immunologicals formed between the NDP kinases and the above-mentioned antibodies.
8. Méthode selon la revendication 7, caractérisée en ce que les anticorps anti-NDP kinases polyclonaux ou monoclonaux sont susceptibles de reconnaître toute ou partie delà revendication la séquence en acides aminés représentée sur la figure 1.8. Method according to claim 7, characterized in that the polyclonal or monoclonal anti-NDP kinase antibodies are capable of recognizing all or part of the claim, the amino acid sequence shown in FIG. 1.
9. Méthode selon la revendication 7, caractérisée en ce que les anticorps anti-NDP kinases polyclonaux ou monoclonaux sont susceptibles de reconnaître toute ou partie des sous-unités A et/ou B des NDP kinases des érythrocytes humains.9. Method according to claim 7, characterized in that the anti-NDP kinase polyclonal or monoclonal antibodies are capable of recognizing all or part of the A and / or B subunits of the NDP kinases of human erythrocytes.
10. Méthode selon la revendication 5, caractérisée en ce qu'elle est réalisée par la mise en présence de l'échantillon biologique étudié avec des sondes d'ADNc spécifiques des NDP kinases, capables de s'hybrider avec les séquences correspondantes des ARNm codant pour les NDP kinases.10. Method according to claim 5, characterized in that it is carried out by bringing together the biological sample studied with cDNA probes specific for NDP kinases, capable of hybridizing with the corresponding sequences of mRNAs coding for NDP kinases.
11. Méthode selon la revendication 10, caractérisée en ce que ces sondes d'ADNc sont constituées d'un enchaînement de nucléotides complémentaires de tout ou partie de l'enchaînement de nucléotides représenté sur la figure 1, ou sont constituées d'un enchaînement de nucléotides susceptibles de s'hybrider avec tout ou partie de l'enchaînement de nucléotides de la figure 1.11. Method according to claim 10, characterized in that these cDNA probes consist of a sequence of nucleotides complementary to all or part of the sequence of nucleotides represented in FIG. 1, or consist of a sequence of nucleotides capable of hybridizing with all or part of the nucleotide sequence in Figure 1.
12. Méthode selon l'une des revendications 1 à il, caractérisée en ce que l'échantillon est une tumeur solide ou un fluide biologique, ou des cellules circulantes12. Method according to one of claims 1 to 11, characterized in that the sample is a solid tumor or a biological fluid, or circulating cells
13. Méthode selon la revendication 12, caractérisée en ce que l'échantillon biologique est utilisé sous forme entière, broyée, en coupe, fractionnée, en particulier sous forme de fractions sub-cellulaires ou, le cas échéant, sous forme de culture cellulaire.13. Method according to claim 12, characterized in that the biological sample is used in whole, crushed, sectioned, fractionated form, in particular in the form of subcellular fractions or, where appropriate, in the form of cell culture.
14. Kit pour la détection in vitro de cellules à fort taux de prolifération selon l'une des revendications 2 à 4 ou 6, caractérisé en ce qu'il renferme :14. Kit for the in vitro detection of cells with a high proliferation rate according to one of claims 2 to 4 or 6, characterized in that it contains:
- des nucleosides diphosphates et triphosphates naturels ou des analogues de synthèse de ces nucléotides, le cas échéant radioactivement marqués, ou sous forme de dérivés 7-thiophosphates de l'ATP ou du GTP,- natural nucleoside diphosphates and triphosphates or analogs of synthesis of these nucleotides, optionally radioactive labeled, or in the form of 7- thiophosphate derivatives of ATP or of GTP,
- un milieu réactionnel approprié pour la réaction enzymatique entre les nucléotides et les NDP kinases, des systèmes enzymatiques couplés permettant la synthèse de produits détectables par colorimétrie et/ou spectrophotométrie, à des dérivés 7-thiophosphates de l'ATP ou de GTP permettant le marquage de l'intermédiaire phosphorylé. - a suitable reaction medium for the enzymatic reaction between nucleotides and NDP kinases, coupled enzymatic systems allowing the synthesis of products detectable by colorimetry and / or spectrophotometry, to 7- thiophosphates derivatives of ATP or of GTP allowing labeling of the phosphorylated intermediate.
15. Kit pour la détection in vitro de cellules à fort taux de prolifération selon l'une des revendications 5, ou 7 à 9, caractérisé en ce qu'il renferme : des anticorps anti-NDP kinases polyclonaux ou monoclonaux , le cas échéant marqués , notamment de manière radioactive ou enzymatique,15. Kit for the in vitro detection of cells with a high proliferation rate according to one of claims 5, or 7 to 9, characterized in that it contains: polyclonal or monoclonal anti-NDP kinase antibodies, where appropriate labeled , in particular radioactive or enzymatic,
- un milieu approprié pour la réaction immunologique entre les susdits anticorps et les NDP kinases,- a medium suitable for the immunological reaction between the above-mentioned antibodies and the NDP kinases,
- des réactifs appropriés pour la détection des complexes immunologiques formés entre les susdits anticorps et les NDP-kinases.- reagents suitable for the detection of the immunological complexes formed between the above-mentioned antibodies and NDP-kinases.
16. Kit pour la détection in vitro de cellules à fort taux de prolifération selon l'une des revendications 10 ou 11, caractérisé en ce qu'il renferme :16. Kit for the in vitro detection of cells with a high proliferation rate according to one of claims 10 or 11, characterized in that it contains:
- des sondes d'ADNc spécifiques des NDP kinases,le cas échéant marquées , notamment de manière radioactive ou enzymatique,- cDNA probes specific for NDP kinases, where appropriate labeled, in particular in a radioactive or enzymatic manner,
- un milieu approprié pour la réaction d'hybridation entre les susdits anticorps et les ARNm codant pour les NDP-kinases,a medium suitable for the hybridization reaction between the above-mentioned antibodies and the mRNAs coding for the NDP-kinases,
- des réactifs appropriés pour la détection des sondes sus-mentionnées hybridées aux ARNm codant pour les NDP kinases.- reagents suitable for the detection of the above-mentioned probes hybridized to mRNAs coding for NDP kinases.
17. Protéine à activité NDP kinase telle qu'obtenue par clonage du gène codant pour la NDP kinase de Dictyostelium discoideum et purification de la protéine synthétisée.17. Protein with NDP kinase activity as obtained by cloning the gene coding for NDP kinase from Dictyostelium discoideum and purification of the synthesized protein.
18. Protéine selon la revendication 17, caractérisée en ce qu'elle est constituée par tout ou partie de l'enchaînement de 155 acides aminés représenté sur la figure 1.18. Protein according to claim 17, characterized in that it consists of all or part of the chain of 155 amino acids shown in Figure 1.
19. Anticorps polyclonaux ou monoclonaux susceptibles de reconnaître toute ou partie de la protéine selon l'une des revendications 18 ou 19, et de se lier immunologiquement avec cette dernière.19. Polyclonal or monoclonal antibodies capable of recognizing all or part of the protein according to one of claims 18 or 19, and to bind immunologically with the latter.
20. Séquence de nucléotides constituée par au moins une partie d'une séquence codant pour une protéine à activité NDP kinase telle que synthétisée par D.discoideum.20. Nucleotide sequence consisting of at least part of a sequence coding for a protein with NDP kinase activity as synthesized by D.discoideum.
21. Séquence de nucléotides selon la revendication 20 caractérisée en ce qu'elle est constituée par tout ou partie de 1'enchaînement de nucléotides représenté sur la figure 1.21. Nucleotide sequence according to claim 20 characterized in that it consists of all or part of the sequence of nucleotides shown in FIG. 1.
22. Séquence de nucléotides capable de s'hybrider avec des gènes codant pour une protéine à activité NDP kinase, notamment avec l'ARNm codant pour cette dernière.22. Nucleotide sequence capable of hybridizing with genes coding for a protein with NDP kinase activity, in particular with the mRNA coding for the latter.
23. Séquence de nucléotides selon la revendication 22 caractérisée en ce qu'elle est susceptible de s'hybrider avec tout ou partie de l'enchaînement de nucléotides représenté sur la figure 1.23. Nucleotide sequence according to claim 22 characterized in that it is capable of hybridizing with all or part of the sequence of nucleotides shown in FIG. 1.
24. Séquence de nucléotides, caractérisée en ce qu'elle est capable de s'hybrider avec une sonde formée à partir de la séquence présentant l'enchaînement de nucléotides représenté sur la figure 1 ou l'enchaînement des nucléotides complémentaires de cette séquence.24. Nucleotide sequence, characterized in that it is capable of hybridizing with a probe formed from the sequence having the sequence of nucleotides shown in FIG. 1 or the sequence of nucleotides complementary to this sequence.
25. Composition pharmaceutique caractérisée en ce qu'elle comprend toute ou partie d'une protéine à activité NDP kinase, en association avec un véhicule physiologiquement acceptable.25. Pharmaceutical composition characterized in that it comprises all or part of a protein with NDP kinase activity, in association with a physiologically acceptable vehicle.
26. Composition pharmaceutique caractérisée en ce qu'elle comprend un polypeptide ou une protéine selon l'une des revendications 17 ou 18, en association avec un véhicule physiologiquement acceptable.26. Pharmaceutical composition characterized in that it comprises a polypeptide or a protein according to one of claims 17 or 18, in association with a physiologically acceptable vehicle.
27. Composition pharmaceutique caractérisée en ce qu'elle comprend des anticorps polyclonaux ou monoclonaux susceptibles de reconnaître toute ou partie d'une protéine à activité NDP kinase, en association avec un véhicule physiologiquement acceptable.27. Pharmaceutical composition characterized in that it comprises polyclonal or monoclonal antibodies capable of recognizing all or part of a protein with NDP kinase activity, in association with a physiologically acceptable vehicle.
28. Composition pharmaceutique caractérisée en ce qu'elle comprend des anticorps polyclonaux ou monoclonaux selon la revendication 20, en association avec un véhicule physiologiquement acceptable.28. Pharmaceutical composition characterized in that it comprises polyclonal or monoclonal antibodies according to claim 20, in association with a physiologically acceptable vehicle.
29. Composition pharmaceutique caractérisée en ce qu'elle comprend une séquence nucléotidique antisens complémentaire de toute ou partie de la séquence nucléotidique codant pour une protéine à activité NDP kinase29. Pharmaceutical composition characterized in that it comprises an antisense nucleotide sequence complementary to all or part of the nucleotide sequence coding for a protein with NDP kinase activity
30. Composition pharmaceutique caractérisée en ce qu'elle comprend une séquence de nucléotides selon l'une des revendications 20 à 24, en association avec un véhicule physiologiquement acceptable. 30. Pharmaceutical composition characterized in that it comprises a nucleotide sequence according to one of claims 20 to 24, in association with a physiologically acceptable vehicle.
EP19900917289 1989-10-31 1990-10-31 Means for detecting highly proliferative cells Withdrawn EP0498849A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
FR8914328 1989-10-31
FR8914328A FR2653781B1 (en) 1989-10-31 1989-10-31 PROCESS FOR THE SYNTHESIS OF ADENOSINE PHOSPHATE DERIVATIVES AND DERIVATIVES OBTAINED THEREFROM.
FR9004754 1990-04-12
FR9004754A FR2660933A1 (en) 1990-04-12 1990-04-12 Means for the detection of highly proliferative cells
US58678490A 1990-09-24 1990-09-24

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DE69033810T2 (en) 1989-10-18 2002-03-28 The Government Of The United States Of America, As Represented By The Secretary National Institute Of Health PRODUCTION AND USE OF THE HUMAN NM23-H2 PROTEIN AND ANTIBODIES AGAINST IT
US5770386A (en) * 1992-05-20 1998-06-23 The United States Of America As Represented By The Department Of Health And Human Services Methods and compositions for increasing the sensitivity of a cell to a DNA damaging agent
CN1301721A (en) * 1999-12-27 2001-07-04 上海博德基因开发有限公司 New polypeptide-photosensitive pigment 10 and polynucleotide codign such polypeptide

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