EP1360293A2

EP1360293A2 - Sequences involved in phenomena of tumour suppression, tumour reversion, apoptosis and/or virus resistance and their use as medicines

Info

Publication number: EP1360293A2
Application number: EP02708409A
Authority: EP
Inventors: Adam Telerman; Robert Amson; Marcel Tuijnder; Laurent Susini
Original assignee: Molecular Engines Laboratories
Current assignee: Molecular Engines Laboratories
Priority date: 2001-02-13
Filing date: 2002-02-13
Publication date: 2003-11-12
Also published as: WO2002064731A3; WO2002064731A2; WO2002064731A9; US20050221303A1; WO2002064731B1; FR2820757A1; JP2004532007A; CA2438219A1

Abstract

The invention concerns in particular novel sequences involved in the molecular pathways of tumour suppression, tumour reversion, apoptosis and/or virus resistance. The invention also concerns the use of said sequences for treatment against cancers, viral diseases, neurodegenerative diseases and in the field of diagnosis and for implementing screening methods for assay of compounds. The invention further concerns methods for detection and/or assay of sequences of the invention or their expression products in a biological sample.

Description

SEQUENCES INVOLVED IN PHENOMENA OF TUMOR SUPPRESSION, TUMOR REVERSION, APOPTOSIS AND / OR VIRUS RESISTANCE AND THEIR USE AS MEDICAMENTS

The present invention relates to the detection of genes involved in the molecular pathways of tumor suppression, tumor reversion, apoptosis and / or resistance to viruses.

The present invention has been made possible by the isolation of cDNAs corresponding to messenger RNAs expressed or repressed during tumor suppression, tumor reversion and / or during the process of apoptosis.

In order to isolate the genes activated or inhibited during tumor reversion, an overall raking of gene expression was carried out in a malignant cell line (U937) and a derived cell line (US4) with suppression of the malignant phenotype. The comparison of the expressed genes (messenger RNA expressed in the two types of cell) made it possible to highlight genes expressed differently, that is to say expressed in one of the cells whereas they are not it in the other (genes can be turned on or off).

It is easy to deduce that these genes are at least involved in the cancerization process, in one case by their absence, and, in the other case, by their presence.

For this differential study, the method used is the method described in 2000 by Brenner et al. (Proc. Natl. Acad, Sci USA, 97 (4), 1665-70).

In order to develop a model, the inventors made the following hypotheses: if it was possible to select, from a tumor which is sensitive to the cytopathic effect of H1 parvovirus, cells which were resistant, then this resistance could be due to a change in their malignant phenotype. This could be demonstrated for the US4 cells selected from the U937 cancer cells. Unlike the parental line U937, the US4 clones (but also US3 which will not be discussed in the present invention) are resistant to the cytopathic effect of the parvovirus H1.

At the molecular level, it has been noted that this suppression of the malignant phenotype goes hand in hand with an activation of the expression of the p21 ^wafl gene and this, independently of the expression of the p53 gene.

The approach to the problem according to the present invention has made it possible to isolate sequences directly linked to several precise functions. Therefore, unlike the random sequencing of TSEs, the sequences are sequences whose function is known because they are involved in the process of suppression of the malignant phenotype, tumor reversion, apoptosis and / or in resistance to viruses.

Tumor reversion is distinguished from tumor suppression by the fact that it encompasses a broader domain than that of tumor suppressor genes. In other words, tumor reversion is achieved by the use of metabolic and / or molecular pathways not limited to the metabolic and molecular pathways in which the tumor suppressor genes are involved.

Thus, the present invention relates in particular to new sequences as well as the use of these sequences in matters of diagnosis and for the implementation of methods for screening for compounds to be tested. The invention also relates to methods for detecting and / or assaying the sequences of the invention or their expression product (s) in a biological sample.

The present invention relates first of all to an isolated nucleotide sequence comprising a nucleotide sequence chosen from the group comprising: a) SEQ LD No. 1 to SEQ DN No. 1020, preferably SEQ LD No. 72, b) a nucleotide sequence of at least 15 consecutive nucleotides of a sequence as defined in a), c) a nucleotide sequence having a percentage identity of at least 80%, after optimal alignment, with a sequence defined in a) or b), d) a nucleotide sequence hybridizing under high stringency conditions with a sequence defined in a) or b), and e) a complementary nucleotide sequence where the RNA sequence corresponding to a sequence as defined in a), b), c) or d).

The nucleotide sequence according to the invention defined in c) has a percentage identity of at least 80% after optimal alignment with a sequence as defined in a) or b) above, preferably at least 90% , most preferably at least 98%.

In the context of the present invention, SEQ LD No. 72 is the preferred nucleotide sequence and corresponds to the TPT1 gene also called TCTP. This gene is notably involved in the phenomenon of tumor reversion and has been the subject of further experiments by the inventors as indicated below.

By nucleotide sequence, nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, terms which will be used interchangeably in the present description, is intended to denote a precise sequence of nucleotides, modified or not, making it possible to define a fragment or region of a nucleic acid, which may or may not contain unnatural nucleotides, and which may correspond to both double-stranded DNA, single-stranded DNA and transcripts of said DNAs. Thus, the nucleic acid sequences according to the invention also include PNA (Peptid

Nucleic Acid), or the like.

The fragments of the nucleotide sequences of the invention comprise at least 15 consecutive nucleotides. Preferably, they comprise at least 20 consecutive nucleotides and even more preferably, they comprise at least 30 consecutive nucleotides.

It should be understood that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, that is to say in the natural state. These are sequences which have been isolated and / or purified, that is to say that they have been taken directly or indirectly, for example by copying, their environment having been at least partially modified. This also means the nucleic acids obtained by chemical synthesis.

By “percentage of identity” between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. The term “best alignment” or “optimal alignment” is intended to denote the alignment for which the percentage of identity determined as below is the highest. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window" to identify and compare the regions. sequence similarity locale. The optimal alignment of the sequences for the comparison can be carried out, besides manually, by means of the algorithm of local homology of Smith and Waterman (1981), by means of the algorithm of local homology of Neddleman and Wunsch (1970 ), using the similarity search method of Pearson and Lipman (1988), using computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI). In order to obtain the optimal alignment, the BLAST program, with the BLOSUM 62 matrix. You can also use the PAM or PAM250 matrices.

The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences, the nucleic acid or amino acid sequence to be compared can include additions or deletions by compared to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.

By nucleic acid sequences having a percentage identity of at least 80%, preferably at least 90%, more preferably at least 98%, after optimal alignment with a reference sequence, is meant the sequences nucleic acids exhibiting, with respect to the reference nucleic acid sequence, certain modifications such as in particular a deletion, a truncation, an elongation, a chimeric fusion, and / or a substitution, in particular pointwise, and whose nucleic sequence has at least 80%, preferably at least 90%, more preferably at least 98%, of identity after optimal alignment with the reference nucleic sequence. Preferably, the specific hybridization or high stringency conditions will be such that they ensure at least 80%, preferably at least 90%, more preferably at least 98% of identity after optimal alignment between one of the two sequences and the complementary sequence of the other.

Hybridization under conditions of high stringency means that the conditions of temperature and ionic strength are chosen in such a way that they allow hybridization to be maintained between two fragments of complementary nucleic acids. By way of illustration - conditions of high stringency of the hybridization step for the purpose of defining the nucleotide sequences described above, are advantageously the following.

DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42 ° C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature depending on the size of the probe (ie: 42 ° C, for a probe of size> 100 nucleotides) followed by 2 washes of 20 minutes at 20 ° C in 2 x SSC + 2% SDS, 1 wash for 20 minutes at 20 ° C in 0.1 x SSC + 0.1% SDS. The last washing is carried out in 0.1 × SSC + 0.1% SDS for 30 minutes at 60 ° C. for a probe of size> 100 nucleotides. The conditions of high stringency hybridization described above for a polynucleotide of defined size, can be adapted by the skilled person for oligonucleotides of larger or smaller size, according to the teaching of Sambrook et al., 1989 .

Among the nucleotide sequences having a percentage identity of at least 80%, preferably at least 90%, more preferably at least 98%, after optimal alignment with the sequences according to the invention, it is preferred also the nucleic sequences variants of the sequences of the invention, or of their fragments, that is to say all of the nucleic sequences corresponding to allelic variants, that is to say individual variations of the sequences of l 'invention.

The term “variant nucleotide sequence” is intended to denote any RNA or cDNA resulting from a mutation and / or variation of a splicing site of the genomic DNA corresponding to the nucleotide sequences of the invention.

The present invention also relates to a polypeptide encoded by a nucleotide sequence in accordance with the invention

By “polypeptide” is meant, within the meaning of the present invention, indifferently designated proteins or peptides. According to a particular embodiment, the polypeptides in accordance with the invention comprise a polypeptide chosen from: a) a polypeptide encoded by a nucleotide sequence in accordance with the invention, b) a polypeptide having at least 80% identity with a polypeptide as defined in a), c) a fragment of at least 5 amino acids of a polypeptide as defined in a) or b), d) a biologically active fragment of a polypeptide as defined in a), b ) or c), and e) a modified polypeptide of a polypeptide as defined in a), b), c) or d).

The evaluation of the percentage of identity is understood after optimal alignment of the sequences concerned. By polypeptide whose amino acid sequence has a percentage identity of at least 80%, preferably at least 90%, more preferably at least 98% after optimal alignment with a reference sequence, intends to designate the polypeptides having certain modifications with respect to the reference polypeptide, such as in particular, one or more deletion (s), truncation (s), an elongation, a chimeric fusion and / or one or more substitution (s).

Among the polypeptides whose amino acid sequence has a percentage identity of at least 80%, preferably at least 90% and more preferably at least 98%, after optimal alignment with a sequence of Reference such as a polypeptide in accordance with the present invention or with one of its fragments, the variant polypeptides encoded by the variant nucleotide sequences as defined above are preferred, in particular the polypeptides whose amino acid sequence has at least a mutation corresponding in particular to a truncation, deletion, substitution and / or addition of at least one residue with respect to the polypeptide sequences of the invention or with one of their fragments. The present invention also relates to a cloning and / or cellular expression vector characterized in that it comprises a nucleotide sequence according to the invention or that it codes for a polypeptide according to the invention. Such a vector can also contain the elements necessary for the expression and optionally for the secretion of the polypeptide in a host cell. Such a host cell is also an object of the present invention.

Vectors comprising promoter and / or regulatory sequences are also part of the present invention. Said vectors preferably comprise a promoter, translation initiation and termination signals, as well as the appropriate regions for regulating transcription. They must be able to be maintained in a stable manner in the cell and can also have specific signals such as to allow the secretion of the translated protein.

These different control signals are chosen according to the cellular host used. To this end, the nucleic acid sequences according to the invention can be inserted into vectors with autonomous replication within the chosen host or integrative vectors of the chosen host.

Among the autonomous replication systems, plasmid or viral type systems are preferably used depending on the host cell, the viral vectors possibly being in particular adenoviruses (5), retroviruses, lentiviruses, poxviruses or herpesviruses. (5a). Those skilled in the art know the technologies that can be used for each of these systems.

When it is desired to integrate the sequence into the chromosomes of the host cell, it is possible to use, for example, systems of the plasmid or viral type; such viruses are, for example, retroviruses (6), or AANs (7).

Advantageously, the vectors according to the invention comprise a sequence ensuring targeting and / or specific tissue expression.

Among the non-viral vectors, naked polynucleotides such as naked ADΝ or naked ARΝ are preferred according to the technique developed by the company VICAL, artificial bacteria chromosomes (BAC, bacterial artificial chromosome), artificial yeast chromosomes ( YAC, yeast artificial chromosome) for expression in yeast, artificial mouse chromosomes (MAC, mouse artificial chromosome) for expression in murine cells and preferably human artificial chromosomes (HAC) for expression in human cells.

Such vectors are prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as for example lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, cell fusion.

The invention furthermore includes host cells, in particular eukaryotic and prokaryotic cells, transformed by the vectors according to the invention as well as transgenic animals, preferably mammals, except humans, comprising one of said cells transformed according to the invention. . These animals can be used as models, for the study of the etiology of inflammatory and / or immune diseases, and in particular of inflammatory diseases of the digestive tract, or for the study of cancers.

Among the cells which can be used for the purposes of the present invention, mention may be made of bacterial cells (8), but also yeast cells (9), as well as animal cells, in particular mammalian cell cultures (10), including Chinese hamster ovary (CHO) cells. Mention may also be made of insect cells in which methods can be used, for example using baculoviruses (11). A preferred cellular host for the expression of the proteins of the invention consists of COS cells.

Among the mammals according to the invention, animals such as rodents, in particular mice, rats or rabbits, which express a polypeptide according to the invention, are preferred.

These transgenic animals are obtained for example by homologous recombination on embryonic stem cells, transfer of these stem cells to embryos, selection of the affected chimeras at the level of the reproductive lines, and growth of said chimeras. The transgenic animals according to the invention can thus overexpress the gene coding for the protein according to the invention, or their homologous gene, or express said gene into which a mutation is introduced. These transgenic animals, in particular mice, are obtained for example by transfection of a copy of this gene under the control of a strong promoter of ubiquitous nature, or selective for a type of tissue, or after viral transcription.

The cells and mammals according to the invention can be used in a method for producing a polypeptide according to the invention, as described below, and can also be used as an analysis model. The transformed cells or mammals as described above can also be used as models in order to study the interactions between the polypeptides according to the invention, and the chemical or protein compounds, involved directly or indirectly in the activities of the polypeptides according to the invention, this in order to study the different mechanisms and interactions involved.

They can in particular be used for the selection of products interacting with the polypeptides according to the invention, or their, as a cofactor, or an inhibitor, in particular a competitive one, or else having an agonist or antagonist activity of the activity of the polypeptides according to the invention. Preferably, said transformed cells or transgenic animals are used as a model, in particular for the selection of products making it possible to combat pathologies linked to an abnormal expression of this gene.

A subject of the present invention is also a monoclonal or polyclonal antibody, a fragment of this antibody or a chimeric antibody capable of specifically recognizing a polypeptide in accordance with the present invention.

The specific monoclonal antibodies can be obtained according to the conventional method of culture of hybridomas well known to those skilled in the art.

The antibodies according to the invention are for example humanized antibodies, Fab or F (ab ') ² fragments. They can also come in immunoconjugates or labeled antibodies to obtain a detectable and / or quantifiable signal.

The antibodies according to the invention but also the immunoconjugates are therefore capable of specifically recognizing a polypeptide according to the present invention.

The specific polyclonal antibodies can be obtained from the serum of an animal immunized against the polypeptides of the invention, in particular produced by genetic recombination or by peptide synthesis, according to the usual procedures. We note in particular the interest of antibodies specifically recognizing the polypeptides, their variants or their immunogenic fragments, according to the invention.

A subject of the invention is also the use of a nucleotide sequence in accordance with the present invention as a probe or primer for the detection, identification, assay and / or amplification of nucleic acid sequences.

According to the invention, the nucleotide sequences which can be used as probe or as primer in methods of detection, identification, assay and / or amplification of nucleic acid sequence, have a minimum size of 15 bases, preferably 20 bases, or better 25 to 30 bases. The probes and primers according to the invention can be labeled directly or indirectly with a radioactive or non-radioactive compound by methods well known to those skilled in the art, in order to obtain a detectable and / or quantifiable signal. The unlabeled nucleic acid sequences according to the invention can be used directly as a probe or primer.

The sequences are generally marked to obtain sequences which can be used for numerous applications. The marking of primers or probes according to the invention is produced by radioactive elements or by non-radioactive molecules.

Among the radioactive isotopes used, mention may be made of ³² P, ³³ P, ³⁵ S, ³ H or ¹²⁵ I. Non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenin, haptens, dyes, luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent, phosphorescent agents.

The nucleotide sequences according to the invention can thus be used as a primer and / or probe in methods using in particular the PCR technique (polymerase chain reaction) (1 Ibis). This technique requires the choice of pairs of oligonucleotide primers framing the fragment which must be amplified. One can, for example, refer to the technique described in U.S. Patent No. 4,683,202. The amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or ion exchange chromatography, and then sequenced. The specificity of the amplification can be controlled by using, as primers, the nucleotide sequences of the invention and, as templates, plasmids containing these sequences or even the amplification products derived therefrom. The amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.

The invention also relates to the nucleic acids capable of being obtained by amplification using primers according to the invention.

Other techniques for amplifying the target nucleic acid can advantageously be used as an alternative to PCR (PCR-like) using pairs of primers of nucleotide sequences according to the invention. By PCR-like is meant to denote all the methods implementing direct or indirect reproductions of the nucleic acid sequences, or in which the labeling systems have been amplified, these techniques are of course known. In general it is the amplification of DNA by a polymerase; when the original sample is an RNA, a reverse transcription should be carried out beforehand. There are currently very many methods allowing this amplification, such as for example the SDA technique (Strand Displacement Amplification) or strand displacement amplification technique (12), the TAS technique (Transcription-based Amplification System) described by (13). , the 3SR (Self-Sustained Sequence Replication) technique described by (14), the NASBA (Nucleic Acid Séquence Based Amplification) technique described by (15) the TMA (Transcription Mediated Amplification) technique, the LCR (Ligase Chain Reaction) technique described by (16), the RCR (Repair Chain) technique

Reaction) described by (17), the CPR (Cycling Probe Reaction) technique described by

(18), the Q-beta-replicase amplification technique described by (19). Some of these techniques have since been perfected.

In the case where the target polynucleotide to be detected is an mRNA, it is advantageous to use, prior to the implementation of an amplification reaction using the primers according to the invention or to the implementation of a method detection using the probes of the invention, an enzyme of reverse transcriptase type in order to obtain a cDNA from lΑRNm contained in the biological sample. The cDNA obtained will then serve as a target for the primers or probes used in the amplification or detection method according to the invention.

The probe hybridization technique can be performed in various ways (20). The most general method consists in immobilizing the nucleic acid extracted from cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and incubating, under well defined conditions, the target nucleic acid immobilized with the probe. After hybridization, the excess probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of radioactivity, fluorescence or enzymatic activity linked to the probe). According to another embodiment of the nucleic acid probes according to the invention, the latter can be used as capture probes. In this case, a probe, called a “capture probe”, is immobilized on a support and is used to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected. thanks to a second probe, called a “detection probe”, marked by an easily detectable element.

The nucleotide sequences according to the invention may moreover be of interest when they are used as antisense nucleotides, that is to say whose structure ensures, by hybridization with the target sequence, an inhibition of expression of the corresponding product. They can also be used as sense nucleotides which, by interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition or an activation of this expression. A subject of the invention is also the use of a nucleotide sequence according to the present invention for the production or the synthesis of a recombinant polypeptide.

The method for producing a polypeptide of the invention in recombinant form, itself included in the present invention, is characterized in that the transformed cells, in particular the cells or mammals of the present invention, are cultivated in conditions allowing the expression of a recombinant polypeptide encoded by a nucleotide sequence according to the invention, and that said recombinant polypeptide is recovered.

Recombinant polypeptides, characterized in that they are capable of being obtained by said production method, also form part of the invention.

The recombinant polypeptides obtained as indicated above can be both in glycosylated and non-glycosylated form and may or may not have the natural tertiary structure. The sequences of the recombinant polypeptides can also be modified in order to improve their solubility, in particular in aqueous solvents.

Such modifications are known to those skilled in the art, such as the deletion of hydrophobic domains or the substitution of hydrophobic amino acids with hydrophilic amino acids.

These polypeptides can be produced from the nucleic acid sequences defined above, according to the techniques for producing recombinant polypeptides known to those skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a cellular host.

An efficient system for producing a recombinant polypeptide requires having a vector and a host cell according to the invention.

These cells can be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, then culturing said cells under conditions allowing replication and / or expression of the transfected nucleotide sequence.

The methods used for the purification of a recombinant polypeptide are known to those skilled in the art. The recombinant polypeptide can be purified from cell lysates and extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using '' specific monoclonal or polyclonal antibodies, etc.

The polypeptides according to the present invention can also be obtained by chemical synthesis using one of the many known peptide syntheses, for example techniques using solid phases (21) or techniques using partial solid phases, by condensation of fragments or by a synthesis in conventional solution. The polypeptides obtained by chemical synthesis and which may contain corresponding unnatural amino acids are also included in the invention.

The present invention also relates to a DNA chip characterized in that it contains at least one nucleotide sequence in accordance with the present invention.

In fact, the nucleotide sequences according to the invention which it is intended to use as a probe or primer for the detection, identification, assay and / or amplification of nucleic acid sequences can be immobilized on a support, covalently or non-covalently, this support being a DNA chip or a high density filter.

By "DNA chip" or "high density filter" means a support on which DNA sequences are fixed, each of which can be identified by its geographic location. These chips or filters differ mainly in their size, the material of the support, and possibly the number of sequences attached to them.

In particular, a synthesis can be carried out in situ by photochemical addressing or by ink jet. Other techniques consist in carrying out an ex situ synthesis and in fixing the probes on the support of the DNA chip by mechanical, electronic or inkjet training. These different methods are well known to those skilled in the art.

The invention also relates to a protein chip comprising a polypeptide or an antibody according to the invention.

Such a protein chip allows the study of interactions between the polypeptides according to the invention and other proteins or chemical compounds and can thus be useful for the screening of compounds interacting with the polypeptides according to the invention.

It is also possible to use the protein chips according to the invention to detect the presence of antibodies directed against the polypeptides according to the invention in the serum of patients to be tested. It is also possible to use a protein chip comprising an antibody according to the invention for this time detecting the presence of polypeptides in the serum of patients capable of being recognized by said antibody. The present invention also relates to the use of a compound chosen from a nucleotide sequence, a polypeptide, a vector, a cell or an antibody according to the invention for the preparation of a medicament.

The pathologies more specifically targeted are viral diseases and diseases characterized by the development of tumor cells or cellular degeneration such as Alzheimer's disease or schizophrenia. Thus, the above drug is intended for the prevention and / or treatment of these diseases. In particular, the disease targeted is cancer.

One of the advantages of the present invention is that it has demonstrated the involvement of a large number of nucleotide sequences in the phenomena of tumor suppression, tumor reversion, apoptosis and / or viral resistance. These sequences are therefore expressed differently when one of these aforementioned processes is started. Consequently, in the presence of a patient whose initiation of one of these processes is suspected or for whom it is desired to verify the absence of such an initiation, it is useful to be able to determine, even to quantify, the expression of one or more sequence (s) in accordance with the invention from a biological sample from said patient. Optionally, the analysis of the expression of one or more of said sequences can be accompanied by a comparison with a reference expression level corresponding to that of a healthy individual. Consequently, the invention therefore also includes a method for the diagnosis and / or prognostic evaluation of a viral disease or characterized by tumor development or cell degeneration comprising the analysis of the expression of at least one sequence of the invention from a biological sample from a patient to be tested. According to a preferred embodiment, said method comprises the following steps:

- isolation of messenger RNA from a biological sample from a patient to be tested, - preparation of complementary cDNA from said messenger RNA,

- optionally, amplification of a portion of complementary DNA corresponding to at least one sequence of the invention, and - detection of the complementary DNA possibly amplified.

In particular, the analysis of the expression of the sequence can be carried out by means of a DNA chip as described above.

Among the sequences of the present invention, some have the characteristic of being receptors expressed on the surface of cells and in order to understand the mechanism thereof within the framework of the processes mentioned above, it is advantageous to search for compounds capable of interact with this receptor, that is to say interact with a polypeptide according to the invention, it is also necessary to provide the secreted protein. This also applies to polypeptides according to the invention corresponding to secreted proteins (on the surface or outside of cells), hormones-like ... Consequently, the present invention also relates to a screening method of compounds capable of binding to a peptide in accordance with the invention and comprising the following steps:

- contacting a polypeptide or a cell according to the invention with a candidate compound, and - detection of the formation of a complex between said candidate compound and said polypeptide or said cell.

A method for screening for compounds may also be advantageous with respect to compounds capable of interacting with a nucleotide sequence according to the invention, or even with the sequences necessary for expression or the regulation of these sequences. Indeed, the compounds are capable of interacting with said sequences with the effect of reducing, inhibiting or on the contrary potentiating the expression of the sequences in question. Such a method comprises the following steps: contacting a nucleotide sequence or a cell according to the invention with a candidate compound, and

- Detection of the formation of a complex between said candidate compound and said nucleotide sequence or said cell.

For reasons mentioned above, it may therefore be advantageous to search for and / or measure, in a biological sample or sample from a patient to be tested, the presence of a nucleotide sequence according to the invention. Such a detection and / or assay method comprises the following steps:

contacting a labeled nucleotide sequence according to the invention with the biological sample to be tested, under the conditions necessary for the formation of a hybrid, and

- Detection and / or assay of the hybrid possibly formed between said nucleotide sequence and the nucleic acid present in said biological sample.

This method can also comprise a step of amplifying the nucleic acid of said biological sample using primers chosen from the nucleotide sequences according to the invention.

In particular, this process can be carried out by means of the DNA chip described above.

A person skilled in the art knows how to implement such a method and can in particular use a reagent kit comprising: a) a nucleotide sequence according to the invention used as a probe, b) the reagents necessary for the implementation of a hybridization reaction between said probe and the nucleic acid of the biological sample, c) the reagents necessary for the detection and / or the assay of the hybrid formed between said probe and the nucleic acid of the biological sample. Such a kit can also contain positive or negative controls to ensure the quality of the results obtained.

Similarly, the detection and / or the assay of a polypeptide according to the invention can also be envisaged in the context of the present invention and this method therefore comprises the following steps:

bringing the biological sample into contact with an antibody labeled according to the invention, and

- detection and / or assay of the complex formed by said polypeptide antibody present in said sample.

Advantageously, this process can be carried out by means of the protein chip as described above. Again, the person skilled in the art knows how to implement such a method and can in particular use a kit of reagents, comprising: a) a monoclonal or polyclonal antibody according to the invention; b) optionally reagents for constituting a medium suitable for the antigen / antibody reaction; c) reagents for the detection of the antigen / antibody complex.

Finally, the subject of the present invention is also a computer-readable medium or a computer medium on which are recorded at least one nucleotide sequence according to claim 1 and / or at least one polypeptide sequence as defined in claim 3 or 4. In particular, this medium is chosen from the group comprising: a) a floppy disk, b) a hard disk, c) a random access memory (RAM), d) a read only memory (ROM), e) a CD-ROM. The invention is not limited to the present description but on the contrary embraces all the variants thereof and will be better understood in the light of the experimental data below.

FIGURES

FIG. 1 represents a tumor growth curve of the U937 and US4 groups in SCID mice. FIG. 2 represents a curve of body weight of the mice carrying a U937 or US4 tumor.

FIG. 3 illustrates: a / the analysis of the expression of the TPT1 / TCTP gene in the U937 / US4.2 lines and in the MCF7 / MCF7 + SIAH1 lines by Northern Blot. The quantities of total transcripts are checked by the GAPDH control; b / Western blot analysis of the expression of the TCTP protein in the U937 / US4.2 model, in the MCF7 / MCF7 + siah model and analysis of the expression of the murine counterpart of TCTP, TCTP MOUSE, in the M1 / LTR6 system after 20 hours of induction at 32 ° C; c / analysis of the expression of TCTP in U937 cells transfected with Panti-sense TPT1 / TCTP. Clones I and LU are compared to the control, U937 cells with the vector alone.

On the right panel, detection of the cleavage of PARP, marker of apoptosis, in clones I and III, U937 cells transfected with Panti-sense TPT1 / TCTP. Jurkat cells treated with an anti-Fas antibody (anti-CD95), which induces apoptosis, serve as a control; d / the characterization of apoptotic cells by double labeling FITC-Annexin V and propidium iodide (PI).

The U937 + control vector cells and the U937 + antisense TPT1 / TCTP cells are simultaneously labeled with annexin V coupled to FITC (which binds to phosphatidylserine in early-stage apoptotic cells) and PI (which only marks necrotic cells whose plasma membrane is damaged). Cytofluorometric analysis makes it possible to count the percentage of apoptotic (Annexin +, PI-), necrotic (Annexin +, PI + or Annexin-, PI +) and intact (Annexin-, PI-) cells; e / the evaluation of apoptosis in cells transfected with Pantis sens TPT1 / TCTP, by in situ labeling of the free 3 'ends of nuclear DNA: TUNEL method ("Terminal deoxyribonucleotidyl transferase- mediated dUTP nick end labeling "). This labeling makes it possible to demonstrate in situ the fragmentation of nuclear DNA resulting from the activation of endonucleases during apoptosis. Apoptotic cells appear green. f7 the study of the in vivo tumorigenicity of U937 cells transfected with: the vector alone (upper curve - 91 tumors / 100), an antisense presenilinl (second curve from the top - 12 tumors / 20), SIAH1 (curve middle - 12 tumors / 20) and a TPTl TCTP antisense, clone I and m (the 2 lower curves - 8 tumors / 20 and 4 tumors / 20). 10 million cells injected into each flank and into each shoulder.

FIG. 4 represents cells cultured on an extracellular tumor matrix (Matrigel). The 184B5 line, mammary epithelial cells transformed by benzo (a) pyrene, is used as a control for the formation of acini. T47D and MCF7 tumor cells form irregular colonies. MCF7 cells transfected with SIAH1 partially recover an organized structure. The MCF7 and T47D cells transfected with Panti-sens TPT1 / TCTP result in cellular reorganization comparable to the normal formation of acini. The cells appearing in green (light spots in the figure) are marked with an anti-E Cadherin antibody coupled to FITC and the nuclei marked with PI appear in red (darker spots). Western blot analysis of TCTP expression in MCF7 and T47D cells transfected with Panti-sense TPT1 / TCTP.

1- DATA RELATING TO U937 and US4 CELLS As seen previously, the present invention uses the parental U937 cells and the “derived” cells US4. In fact, US4 cells and US3 cells (which are not discussed in the context of the present invention) share certain characteristics. Their method of production and their properties are reported below. Selection and characterization of US3 and US4 cells

The U937 cells were subjected to two series of limited dilution until a single clonal population was obtained. These cells were infected with the parvovirus H1. The cytopathic effect of the virus creates a massive cell death sparing two resistant clones that are US3 and US4 after three months of continuous culture. Cell survival is defined as the relative number of viable cells in the culture infected with the Hl virus compared to the untreated culture, as measured four days after reinfection. To measure the tumorigenicity, 10 ⁷ cells of U937, US3 or US4 were injected subcutaneously in scid / scid mice (4 or 5 weeks old). The tumorigenicity is expressed by the number of tumors developed by the mice in the two months following the injection.

The approach was as follows: from clonal populations of malignant cells, subclones were derived with a suppressed tumorigenic phenotype. This selection made by means of the parvovirus H-1 is carried out by the elimination of the tumor cells which are preferentially killed while sparing the normal cells. Selection of cells resistant to the cytopathic effect of parvovirus H-1 outside of a sensitive tumor can give rise to cells which have a reduced malignant phenotype.

On this basis, a clonal population of U937 cells has been isolated, these cells are sensitive to the cytopathic effect of the parvovirus H1 and the clones US3 and US4 are resistant to the virus. The clones US3 and US4 have a strong tumorigenic phenotype suppressed while the parental U937 cells develop tumors in 80% of the scid / scid mice having been infected with parvovirus, the US3 cells form a single tumor and the US4 cells develop a tumor for 20 inoculations with 10 ⁷ cells. The results are collated in the table below.

Resistance to H-1 parvovirus and tumorigenicity of U937 cells. US3 and US4

2- MATERIALS AND METHODS

This involves comparing the growth of subcutaneous tumors in SCID mice induced by subcutaneous injection of transfected human leukemic cell lines U937 and US4.

A. Leukemic cell lines and culture conditions

All cells injected from the U937 (ATCC) and US4 cell lines were provided as cell suspensions in vials supplemented with RPMI-1640 culture medium supplemented with 2mM L-glutamine, 10% fetal bovine serum and gentamycin.

The U937 cell line is a human CD4 + monocytic cell line derived from a patient with diffuse histiocytic lymphoma (1).

The cells were counted in a hemocytometer and their viability was tested with exclusions of 0.25% trypan blue dyes. Viability was 95.5% and 90.5% respectively for U937 cells and US4 cells. The U937 and US4 cells were centrifuged and then resuspended in RPMI medium before being injected into SCID mice.

B. The animals

10 healthy SCID female mice (CB17 / IcrHsd), 31 weeks old and weighing between 20 and 25 g were supplied by Harlan France (Gannat, France). The animals were observed for 7 days in a specific room of the Applicant which is the care unit for animals without specific pathogens (SPF for specific-pathogen-free) before their treatment. The animal care unit (INRA, Dijon, France) is authorized by the French Ministers of Agriculture and Research (approval n ° A21100). Animal experiments are carried out in accordance with the European ethical guidelines on animal experiments (2) and the British directives on animal welfare in the context of experimental neoplasia (3).

B.l. Environment

The animals were kept in rooms under controlled conditions of temperature (24 ± 1 ° C), humidity (55 ± 1%), period of light (12 h of light / 12 h of night) and renewal air. The animals were kept under SFP conditions and the room temperature and humidity were continuously monitored. The ventilation system has been programmed to give rise to 14 air changes per hour without recirculation. Fresh air from the outside passes through a series of filters before being distributed regularly in each room. High pressure (2 mm) was maintained in the experimental rooms to prevent contamination or spread of pathogens within a colony of mice. All personnel working under SPF conditions followed the specific directives with regard to hygiene and clothing when they entered the breeding area. B.2. livestock

The animals were housed in polycarbonate cages (UAR, Epinay sur Orge, France) which were equipped to provide them with food and water. The standard size of the cages used is 637 cm ² for 10 mice according to internal standard operating procedures. The animal bedding made of sterile wood shavings (PSU) and replaced twice a week.

B.3. Food and drink

The animal feed was purchased from Extralabo (Provins, France). The feed was supplied ad libitum and was placed on the metal cover at the top of the cage. Water was also supplied ad libitum from water bottles fitted with rubber taps. The water bottles were washed, sterilized and replaced once a week. The water supply was sterilized by filtration with an absolute filter of 0.2 μm. B.4. Identification of the animal and the cage

After random distribution, the animals were identified with 2 different numbers engraved on the two ears. Each cage has been marked with a specific code.

C- Experimental data and treatments

Cl. Induction of tumors in SCID mice

Before the cell injection, the SCID mice were randomly divided into 2 groups, at a rate of 5 mice per group. 10 ⁷ US4 or U937 tumor cells in 0.2 ml of RPMI medium were inoculated subcutaneously at time 0 for each injection site in SCID mice. Each animal received 4 injections of tumor cells located in different areas. One in each side and one in each shoulder.

C.2. Collection of tumors When the tumors reached a volume of 1500 mm ³ , the mice were killed and the tumors were collected, weighed and frozen in liquid nitrogen, stored at -80 ° C. and then labeled specifically.

C.3. Mouse control

Isoflurane forene (Minerve, Bondouble, France) was used to anesthetize animals before injecting cells for sacrifice. After the injection of tumor cells, the mice were observed for 5 hours. Viability, behavior, body weight of mice and growth of the subcutaneous tumor were recorded twice a week.

During the duration of the experiment, the animals were killed under anesthesia with isoflurane by cervical dislocation if one of the following signs appeared:

- sign of suffering (cachexia, weakness, difficulty moving or eating),

- tumor growth up to 10% of body weight,

- tumor ulceration and persistent exposure,

- position of the tumor interfering with movement and / or food, - weight loss of 20% for three consecutive days.

An autopsy of each animal was performed to detect the presence of possible metastases or morphological anomalies.

D- Presentation of data P.1. Survival parameters

The calculation for median and mean survival time was expressed as follows:

Average survival time = S. / (S ₂ -NT) With: SI = the sum of daily survivors from day 0 until the end of the experiment (without survivors "not taken into account" *) S2 = the number of animals at the origin NT = the number of animals "not taken into consideration" * * "not taken into consideration": these are animals with tumors smaller than the predetermined limit considered as a result of a tumor implantation defect.

D.2. Tumor inhibition system

The tumor size was measured twice a week with a compass and the tumor volume (in mm ³ ) will be estimated according to the formula: (length x width ² ) ^ (4). The experiments were stopped when the tumor sizes of the mice reached 1,500 mm ³ .

- After the sacrifice, the tumors were excised and weighed.

- The curve of tumor growth in the US4 and U937 groups was plotted using the average of the tumor volumes.

- The tumor doubling time of the US4 and U937 groups was defined as the period required to reach an average tumor volume of 200% during the growth period.

- The specific growth period over one or two doubling times (TD) from cell injections is defined as follows: specific growth period = (TD US4- TD U937) / TD U937 - the growth period was calculated as the difference in the median growth time of the US4 group and the U937 group to reach the same tumor size.

D.3. Statistical tests

All the statistical analyzes were carried out with StatView® software (Abacus Concept, Berkeleley, USA). Statistical analyzes of changes in average body weight, tumor doubling time and time to "V" was performed using the Bonferroni / Dunn test. A p value <0.05 was considered significant. All groups were compared with themselves. E- Results

The curves of tumor volumes and average body weight are shown in Figures 1 and 2 respectively. No significant loss of body weight of SCID mice from the 2 groups was observed between day 8 and day 19.

A significant difference was observed concerning the time to reach "V" between the 2 groups of SCID mice while no significant difference was observed for the change in average body weight (day 19-day 8) and for the time of dubbing.

The autopsies carried out did not show the presence of metastases or development of suspicious nodosity. Tumors were collected from animals sacrificed after anesthesia and cervical dislocation. The tumors were immediately put into tubes, frozen in liquid azole and stored at -80 ° C. The excised tumors had an ovoid shape, a moderate consistency and a pinkish color. Interactions of the tumor with its environment (skin and muscle tissue has been limited and superficial).

F- Conclusions The US4 cell line showed a significantly lower uptake rate compared to the U937 cell line in SCID mice.

The growth retardation between the US4 and U937 tumors was 23.5 days and the doubling time was equivalent.

3- INVOLVEMENT OF TPTl / TCTP IN THE REVERSION

TUMOR

In order to study the implication of TPTl / TCTP (Translationally controlled Tumor Protein encoding the Histamine Releasing Factor) in tumor reversion, gene expression (Figure 3a) and protein expression (Figure 3b) in different cellular models of tumor reversion: U937 / US4.2, MCF7 / MCF7 + SIAH1 and M1 / LTR6, were analyzed. The level of expression of TPT1 / TCTP is greatly reduced in the returning cells compared to the tumor cells. To address the physiological aspect of this decrease, the U937 tumor cells were transfected with Panti-sens TPT1 / TCTP. After verifying the drop in expression of TPT1 / TCTP (Figure 3c, left panel), the inventors detected its consequences in apoptosis by three different methods: detection of the cleavage of PARP, labeling with PAnnexinV and method TUNEL ( respectively figure 3 c, right panel; figure 3d; figure 3 e)

The decrease in expression of TPT1 / TCTP in cultured tumor cells increases apoptosis.

The inventors then examined in vivo the physiological consequences of this loss of function of TPT1 / TCTP. We injected U937 cells transfected with Panti-sens TPT1 / TCTP into mice and observed a drastic reduction in the number of tumors formed when the expression of TPT1 / TCTP is reduced (Figure 3f).

In parallel with these results, the reduction of TPTl / TCTP, obtained by transfection of double strand RNA specific for TPTl / TCTP (siRNA), in MCF7 and T47D cells, cultured on an extracellular matrix, leads to cellular reorganization into comparable acini. to a normal structure (Figure 4).

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Claims

1. Isolated nucleotide sequence comprising a nucleotide sequence chosen from the group comprising: a) SEQ LD No. 72, b) a nucleotide sequence of at least 15 consecutive nucleotides of a sequence as defined in a), c) a sequence nucleotide having a percentage identity of at least 80%, after optimal alignment, with a sequence defined in a) or b), d) a nucleotide sequence hybridizing under conditions of high stringency with a sequence defined in a) or b), and e) a complementary nucleotide sequence or the RNA sequence corresponding to a sequence as defined in a), b), c) or d).

2. Nucleotide sequence according to claim 1, characterized in that its expression is involved during tumor suppression, tumor reversion, apoptosis and / or viral resistance.

3. A polypeptide encoded by a nucleotide sequence according to claim 1.

4. Polypeptide according to claim 3, characterized in that it comprises a polypeptide chosen from: a) a polypeptide according to claim 3, b) a polypeptide having at least 80% identity with a polypeptide as defined in a) , c) a fragment of at least 5 consecutive amino acids of a polypeptide as defined in a) or b), d) a biologically active fragment of a polypeptide as defined in a), b) or c), and e) a modified polypeptide of a polypeptide as defined in a), b), c) or d).

5. A cloning and / or cell expression vector characterized in that it comprises a nucleotide sequence according to claim 1 or coding for a polypeptide according to claim 3 or 4.

6. Vector according to claim 5, characterized in that it is a viral vector chosen from an adenovirus, a retrovirus, a herpes virus or a poxvirus.

7. Vector according to claim 5, characterized in that it is a naked nucleic acid vector.

8. Vector according to one of claims 5 to 7, characterized in that it comprises a sequence ensuring targeting and / or specific tissue expression.

9. Host cell transformed with an expression vector according to one of claims 5 to 8.

10. Animal, preferably a mammal except man, characterized in that it comprises a cell according to claim 9.

11. Monoclonal or polyclonal antibody, fragment of this antibody or chimeric antibody, characterized in that it is capable of specifically recognizing a polypeptide according to one of claims 3 and 4.

12. Use of a nucleotide sequence according to claim 1 as a probe or primer for the detection, identification, assay and / or amplification of nucleic acid sequences.

13. Use in vitro of a nucleotide sequence according to claim 1, as sense or antisense nucleotide.

14. Use of a nucleotide sequence according to claim 1 for the production of a recombinant polypeptide.

15. A method of obtaining a recombinant polypeptide characterized in that a cell according to claim 9 is used under conditions allowing the expression of said polypeptide and that said recombinant polypeptide is recovered.

16. DNA chip characterized in that it contains at least one nucleotide sequence according to claim 1.

17. Protein chip characterized in that it contains a polypeptide according to claim 3 or 4 or an antibody according to claim 11.

18. Use of a compound chosen from: a) a nucleotide sequence according to claim 1, b) a polypeptide according to claim 3 or 4, c) a vector according to one of claims 5 to 8, d) a cell according to claim 9, e) an antibody according to claim 11, for the preparation of a medicament

19. Use according to claim 18, characterized in that the medicament is intended for the prevention and / or treatment of viral diseases or characterized by the development of tumor cells or cellular degeneration.

20. Use according to claim 19, characterized in that the disease is cancer.

21. Method for the diagnosis and / or prognostic evaluation of a viral disease or characterized by tumor development or cell degeneration comprising analysis of the expression of at least one sequence corresponding to SEQ LD N ° 72, from a biological sample from a patient to be tested.

22. Method of diagnosis and / or prognostic evaluation according to claim 21, comprising the following steps:

- isolation of messenger RNA from a biological sample from a patient to be tested,

- preparation of complementary DNA from said messenger RNA,

- optionally, amplification of portions of complementary DNA corresponding to at least one sequence corresponding to SEQ LD No. 72,

- detection of complementary DNA, possibly amplified.

23. The method of diagnosis and / or prognostic evaluation according to claim 22, in which the analysis of the expression of the sequence is carried out by means of a DNA chip according to claim 16.

24. A method of screening for compounds capable of interacting with a nucleotide sequence according to claim 1, characterized in that it comprises the following steps: - bringing into contact a nucleotide sequence according to claim 1, or a cell according to claim 9 with a candidate compound, and - Detection of the formation of a complex between said candidate compound and said nucleotide sequence or said cell.

25. A method of screening for compounds capable of binding to a polypeptide according to claim 3 or 4, characterized in that it comprises the following steps:

contacting a polypeptide according to claim 3 or 4 or a cell according to claim 9 with a candidate compound, and

- Detection of the formation of a complex between said candidate compound and said polypeptide or said cell.

26. A method of detecting and / or assaying a nucleotide sequence according to claim 1 in a biological sample, characterized in that it comprises the following steps: - contacting a nucleotide sequence according to claim 1 marked with the biological sample to be tested, under the conditions necessary for the formation of a hybrid,

27. Detection and / or assay method according to claim 26, characterized in that it comprises a step of amplification of the nucleic acid of said biological sample using primers chosen from the nucleotide sequences as defined in claim 1.

28. Detection and / or assay method according to claim 26 or 27, characterized in that it is carried out by means of the DNA chip as defined in claim 16.

29. Method for detecting and / or assaying a polypeptide as defined in claim 3 or 4 in a biological sample, characterized in that it comprises the following steps:

bringing the biological sample into contact with a labeled antibody as defined in claim 11,

- Detection and / or assay of the complex formed between said antibody and the polypeptide present in said sample.

30. A method of detecting and / or assaying a polypeptide according to claim 29, characterized in that it is carried out by means of the protein chip as defined in claim 17.