FR2804690A1 - NEW PROTEIN FAMILIES, NAMED ATIP, NUCLEIC SEQUENCES ENCODING SAID PROTEINS AND THEIR APPLICATIONS - Google Patents

Abstract

New family of human proteins (hATIP), interacting with the angiotensin AT2 receptor. It and having an anti-oncogene function. Said family of proteins is therefore useful for the diagnosis of cancerous and precancerous states and the treatment of cancerous states. Gene encoding said proteins, fragments of said gene and their applications for the detection and expression of said proteins. Antibodies directed against said proteins and also to methods of detecting and / or screening for a precancerous or cancerous state using said proteins, said antibodies and / or different nucleic acid sequences.

Description

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NEW FAMILY OF PROTEINS, NAMED ATIP, NUCLEIC SEQUENCES ENCODING SAID PROTEINS AND THEIR APPLICATIONS.

 The present invention relates to a new family of human proteins (hATIP), interacting with the angiotensin II AT2 receptor and having an anti-oncogenic function. Said family of proteins is therefore useful for the diagnosis of cancerous and precancerous states and the treatment of cancerous states.

 The present invention also relates to the gene coding for said proteins as well as to transcripts and cDNA molecules coding for said proteins, to fragments of these different nucleic acid molecules and to their applications for detection and expression. of said proteins.

 The present invention also relates to nucleic sequences (genomic DNA, transcribed and cDNA) or protein of the hATIP family, having an alteration or a functional mutation.

 The present invention also relates to the various hATIP proteins and to their applications.

 The present invention further relates to antibodies directed against said proteins as well as to methods of detecting and / or screening for a precancerous or cancerous state using said proteins of said antibodies and / or of the different sequences of 'nucleic acids.

 The angiotensin II AT2 receptor is a receptor with seven transmembrane domains, which thus belongs to the family of receptors coupled to G proteins. Its role during tissue development and regeneration has recently been demonstrated (Nouet et al., Trends Endocrinol.

Metabol., 2000,11,1-6). Mice whose AT2 gene has been disabled have abnormalities in the development of the urinary tract due to a defect in apoptosis of the mesenchymal cells (Nishimura et al., Molecular Cell, 1999,3, 1-10). In fact, this receptor has an effect in many tissues

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 anti-proliferative and pro-apoptotic by means of the activation of protein tyrosine phosphatases, among which the tyrosine phosphatase SHP-1 (Bedecs et al., Biochem. J., 1997,325, 449-454; Lehtonen et al., Mol. Endocrinol., 1999, 13, 1051-1060). Very recently, an effect of the AT2 receptor on cell differentiation and migration has been demonstrated (Côté et al., J.

Biol. Chem., 1999,274,44,31686-31692).

 All these effects (growth inhibition, apoptosis, differentiation and cell migration) are in agreement with the predominant role played by the AT2 receptor in the growth and remodeling of tissues. However, the intracellular mechanisms associated with the activation of this receptor and its growth-inhibiting effects remain poorly understood.

 To progress in understanding the intracellular signaling pathways of the AT2 receptor, proteins interacting with the intracytoplasmic portions of this receptor have been sought.

 Initial studies, undertaken using the double hybrid cloning method in yeast, have led to the characterization of a human protein capable of binding to the AT2 receptor (SEQ ID NO: 54), which has been the subject of PCT / FR99 / 01908.

 This protein, now called hATIPl, interacts with the C-terminal end of the AT2 receptor, but not with those of other receptors of the same family, such as the AT1 receptors, (32-adrenergic or bradykinin B2.

 By interacting with the AT2 receptor, the hATIPl protein plays a role in the signaling pathways of the AT2 receptor and contributes to attenuating the cell proliferation induced by growth factors.

 Continuing their work, some of the inventors have now effectively identified new nucleic acid sequences coding for proteins homologous to hATIP1 (family hATIP) and interacting with the AT2 receptor as well as genomic sequences comprising the hATIP gene or at least most of said uncomfortable; They also have

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 identified this gene as a tumor suppressor gene. The invention also relates to the use of these sequences for the detection of abnormalities in the expression or of the sequence of the proteins of the hATIP family in certain proliferative lesions and for the treatment of cancer cells including all or part of the hATIP a gene. been altered or deleted.

 A large number of studies describe the presence of one or more tumor suppressor genes, located on the small arm of chromosome 8, in position 8p21.3-p22. All or part of the small arm of chromosome 8 is frequently deleted or mutated in tumors of the colon, liver, lung, prostate, breast, ovaries, ENT sphere and bladder (Miyakawa et al., Oncol. Rep., 1999.6, 785-789; Radford et al., Genomics, 1999.60, 1-11; Yoshimoto et al., Cancer, 1999.85, 447-452). The complete sequence of genomic DNA (1.2 million nucleotides) contained in the 8p21.3-p22 segment has recently been published in the databases (Genbank n AB020859 to n AB020868). However, the determination of this sequence neither makes it possible to identify the sequences effectively coding nor thus to determine the identity of the tumor suppressor gene (s).

 The characterization of the hATIP protein family has enabled the inventors to determine the structure and organization into introns / exons of the gene coding for these proteins, located in the chromosomal region 8p21.3-p22.

 The human hATIP gene is organized into at least 17 exons, distributed over more than 112 kilobases of genomic DNA; some of these exons can be used alternately, which leads to transcription of different mRNA molecules. Due to this alternative splicing, these different mRNA molecules have different translation initiating ATG codons and therefore code for proteins of different sequences; the sequences coding for different isoforms of proteins of the hATIP family (hATIP2, hATIP3, hATIP4 and hATIP5) have thus been identified.

 While the different proteins of this family diverge

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 in their N-terminal part, they present in their C-terminal part, a common zone which characterizes this family and which includes the zone of interaction with the AT2 receptor.

 The subject of the present invention is therefore an isolated cDNA sequence, characterized in that it is selected from the group consisting of: (a) the nucleic acid sequences coding for an hATIP protein, comprising at least one fragment common to all the hATIP proteins of sequence SEQ ID NO: 11, which corresponds to the region of interaction with the AT2 receptor, which nucleic acid sequence does not include the sequence SEQ ID NO: 55; excluding the GENBANK sequences n AB033114 and n AL096842; (b) allelic variants of the nucleic acid sequence as defined in (a) (c) sequences complementary to the nucleic acid sequences, as defined in (a) and in (b); and (d) homologous sequences from any mammal.

 Within the meaning of the present invention, the term “homologous sequence” means a sequence whose identity with the corresponding human sequence (after alignment of sequences) varies from 80 to 99%.

 According to an advantageous embodiment of said cDNA sequence, it comprises a sequence selected from the group consisting of the sequence SEQ ID NO: 9, a sequence corresponding to positions 364-714 of SEQ ID NO: 9 and a sequence which hybridizes under stringent conditions (under the conditions described in Sambrook et al., 1989) with the sequence SEQ ID NO: 9 and its fragments.

 The sequence SEQ ID NO: 9 corresponds to a nucleic sequence segment common to all the sequences coding for an ATIP protein, which segment includes the sequence coding for the interaction region

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 with the AT2 receptor.

 The cDNA sequences according to the present invention correspond to the different mRNAs, obtained by alternative splicing and coding respectively for the isoforms hATIP2, hATIP3, hATIP4 and hATIP5.

 More precisely, - the sequence coding for the hATIP2 protein has the sequence SEQ ID NO: 1 - the sequence coding for the hATIP3 protein has the sequence SEQ ID NO: 3 - the sequence coding for the hATIP4 protein has the sequence SEQ ID NO: 5 and - the sequence coding for the hATIP5 protein has the sequence SEQ ID NO: 7.

 These different cDNA sequences correspond to alternative splices of the hATIP gene; they make it possible to reconstitute the organization of the gene into exons and introns and therefore to define the coding regions.

 The present invention also relates to a genomic nucleic acid segment corresponding to the hATIP gene coding for an hATIP protein, which sequence is characterized in that it is selected from the group consisting of: (a) a sequence which corresponds to the concatenation of positions 12,636 to 1 of the Genbank sequence n AB020865 and of positions 100,000 to 688 of the Genbank sequence of accession AB020864, which codes at least for the proteins hATIPl, hATIP2, hATIP3, hATIP4 and hATIP5 (SEQ ID NO : 2, 4, 6, 8 and 54) and which comprises at least 17 coding exons whose reading is done on the complementary reverse strand of said sequences n AB020865 and n AB020864; excluding said sequences n AB020865 and n AB020864; (b) allelic variants of the nucleic acid sequence

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as defined in (a); (c) mutants of the nucleic acid sequence as defined in (a) or in (b) which comprise at least the substitution of a nucleotide at the level of at least one of said exons; preferably, said substitution is found in at least one hATIP gene allele in at least one tumor cell line and is not found in a population of
100 alleles of the hATIP gene from normal cells; (d) sequences complementary to the nucleic acid sequences, as defined in (a) and in (b) and (e) homologous sequences originating from any mammal.

 More precisely, said exons are distributed as follows over said genomic sequence (see FIG. 4): - exon 1 (sequence SEQ ID NO: 36): it comprises at least 2032 bp and corresponds to positions 12651 to 10620 of sequence AB020865 (segment 8/11); it codes for the N-terminal part of hATIP3, the initiator ATG codon of which is in position 86 of the sequence SEQ ID NO: 36 (or 12550 of AB020865). One can observe, in the sequences originating from cells of different origins, a C / T substitution in position 1973 (SEQ ID NO: 36) or 10664 (AB020865); - exon 2 (sequence SEQ ID NO: 37): it comprises 196 bp and corresponds to positions 702 to 507 of AB020865 (segment 8/11); - exon 3 (sequence SEQ ID NO: 38): it comprises 61 bp and corresponds to positions 81060 to 81000 of the sequence AB020864 (segment 7/11); it is present in the cDNA of hATIP2 (uterus) and absent in the cDNA of hATIP3 (brain); it can therefore be spliced alternately and characterizes the hATIP2 transcript; - exon 4 (sequence SEQ ID NO: 39): it comprises 162 bp and corresponds to positions 80731 to 80570 of AB020864 (segment 7/11).

 - exon 4 bis (sequence SEQ ID NO: 40): it comprises at least

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94 bp and corresponds to positions 78701 to 78608 of AB020864 (segment 7); perhaps the beginning of the exon is missing; this exon is contained only in the sequence encoding hATIP5; it can therefore be spliced alternately and characterizes the hATIP5 transcript; - exon 5 (sequence SEQ ID NO: 41): it comprises 135 bp and corresponds to positions 72800 to 72666 of AB020864 (segment 7/11); it contains the initiator ATG codon of hATIP2 at position 72684 of AB020864 or
117 of SEQ ID NO: 41).

 - exon 6 (sequence SEQ ID NO: 42): it comprises 39 bp and corresponds to positions 70152 to 70114 of AB020864 (segment 7/11).

 - exon 7 (sequence SEQ ID NO: 43): it comprises 413 bp and corresponds to positions 54567 to 54155 of AB020864 (segment 7/11); this exon is used exclusively by hATIPl (# alternative splicing). ; it contains the ATA codon initiating hATIPl at position 54275.

 - exon 8 (sequence SEQ ID NO: 44): it comprises 215 bp and corresponds to positions 41450 to 41236 of AB020864 (segment 7/11); one can observe, in position 41342 of AB020864, an A / C substitution, which gives a Lys / Thr mutation, in particular in certain tumors.

 - exon 9 (sequence SEQ ID NO: 45): it comprises 67 bp and corresponds to positions 32194 to 32128 of AB020864 (segment 7/11).

 - exon 10 (sequence SEQ ID NO: 46): it comprises 203 bp and corresponds to positions 12951 to 12749 of AB020864 (segment 7/11).

 - exon 11 (sequence SEQ ID NO: 47): it comprises 106 bp and corresponds to positions 11550 to 11445 of AB020864 (segment 7/11).

 - exon 12 (sequence SEQ ID NO: 48): it comprises 74 bp and corresponds to positions 10382 to 10309 of AB020864 (segment 7/11).

 - exon 13 (sequence SEQ ID NO: 49): it comprises 96 bp and corresponds to positions 10165 to 10070 of AB020864 (segment 7/11); a G / C substitution is observed, at position 10141 of AB020864, which gives a Glu / Gln mutation in certain tumors.

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 - exon 14 (sequence SEQ ID NO: 50): it comprises 117 bp and corresponds to positions 6845 to 6729 of AB020864 (segment 7/11).

 - exon 15 (sequence SEQ ID NO: 51): it comprises 98 bp and corresponds to positions 3961 to 3864 of AB020864 (segment 7/11) and - exon 16 (sequence SEQ ID NO: 52): it comprises 2333 bp and corresponds at positions 3021 to 689 of AB020864 (segment 7/11); it contains the stop codon common to all the hATIP proteins (TGA in position 117 of the sequence SEQ ID NO: 52).

 The exons used by all of the isoforms of hATIP are exons 8 to 16.

 The present invention also relates to fragments of said genomic sequences, characterized in that they consist of one of the exons comprising at its 5 'end and / or at its 3' end, an intron segment comprising of 1 at 300 bp; said fragment preferably comprises between 15 and 50 bp, and is included in said intron segment of the genomic sequence which can be located at a distance of 1 to 200 bp upstream and / or downstream of said exons.

 More precisely, said fragments are selected from the sequences SEQ ID NO: 56-72.

 In these sequences, the border between intron and exon should not be considered as precise to the nearest nucleotide.

 The present invention also includes fragments of the nucleic acid sequences as defined above and their complementary sequences (including the transcripts).

 Preferably: - when these fragments comprise between 50 and 1000 bp, they are useful as probes; - When these fragments comprise between 15 and 50 bp, they are useful as primers.

 Among said fragments, mention may in particular be made of frag-

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following:
1. probes:. Probe B1260 (sequence SEQ ID NO: 18): hybridizes all the sequences coding for an hATIP; extends over exons 11 to 16; it is obtained by RT-PCR with the oligonucleotides B1249 (SEQ ID NO: 21) / B1251 (SEQ
ID NO: 22); it makes it possible to detect the hATIP gene and the various forms of the hATIP mRNA by hybridization of the genomic DNA or of the human mRNA under stringent hybridization conditions.

 . Probe B1298 (sequence SEQ ID NO: 19): specific for hATIP2, hATIP3, hATIP4 and hATIP5; extends over exons 4 to 6 with the exception of exon 4bis; it is obtained by RT-PCR with the oligonucleotides B1264 (SEQ ID NO: 23) / B1265 (SEQ ID NO: 24); it makes it possible to identify the sequences coding for hATIP2, hATIP 3, hATIP 4 and hATIP5, but does not hybridize with the hATIP1 sequence, even under slightly stringent hybridization conditions.

 . SEQ ID NO: 20 sequence probe, specific for hATIPl; includes only exon 7; it is obtained by PCR on genomic DNA or by RT-PCR with the oligonucleotides B1262 (SEQ ID NO: 25) / B1301 (SEQ ID NO: 26).

 2. fragments useful for amplifying a segment coding for an hATIP: a) primers for the amplification of the B1260 probe (common to all hATIPs) (sequence SEQ ID NO: 18): - oligonucleotide B1249: 23 -sense ( SEQ ID NO: 21), located in exon 11 (SEQ ID NO: 47): positions 11501 to 11523 of AB020864 and - oligonucleotide B1251: antisense (SEQ ID NO: 22), located in exon 16 (SEQ ID NO: 52): positions 2711 to 2734 of AB020864. b) primers for the amplification of the B1298 probe (specific for hATIP2, hATIP3, hATIP4 and hATIP5) (SEQ ID NO: 19): - oligonucleotide B1264: 21-mer sense (SEQ ID NO: 23), located in the exon 4 (SEQ ID NO: 39): positions 80708-80728 of AB020864 and

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 - oligonucleotide B1265: antisense (SEQ ID NO: 24), located in exon 6 (SEQ ID NO: 42): positions 70111-70129 of AB020864. c) primers for the amplification of the sequence SEQ ID NO: 20, specific for hATIPl (amplification of exon 7) - oligonucleotide B1262: 21-mer sense (SEQ ID NO: 25), located in exon 7 (SEQ ID NO: 43): positions 54512-54532 of AB020864 and - oligonucleotide B1301: 19-mer antisense (SEQ ID NO: 26), located in exon 7 (SEQ ID NO: 43): positions 54158-54177 from AB020864.

 3. fragments useful for detecting a polymorphism or a mutation appearing in cancer cells: a) amplification of exon 8 in order to detect the Lys / Thr mutation (AA 56 of the sequence SEQ ID NO: 2): - oligonucleotide B1318: 22-mer sens (SEQ ID NO: 27), located in the intron preceding exon 8 (SEQ ID NO: 64): positions 41584-41605 of AB020864; - oligonucleotide B1319: 22-antisense (SEQ ID NO: 28), located in the intron following exon 8 (SEQ ID NO: 64): positions 41221-41242 of AB020864.

 This pair of oligonucleotides produced by amplification of genomic DNA, a 385 bp fragment, in which an A / C substitution at position 41342 of AB020864 generates a restriction site for the NIaIII enzyme: fragments of 258 and 127 bp, if the DNA is mutated, whereas there is no cleavage in the wild-type DNA: a single fragment of 385 bp is obtained in this case. b) amplification of exon 13 in order to detect the Glu / Gln mutation (AA 250 of the sequence SEQ ID NO: 2): - oligonucleotide B1308: 22-mer sense (SEQ ID NO: 29), located in the intron preceding exon 13 (SEQ ID NO: 69): positions 10205-10226 of AB020864; and - oligonucleotide B1309: 22-mer antisense (SEQ ID NO: 30),

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 located in the intron following exon 13 (SEQ ID NO: 69): positions 9894-9915 of AB020864.

This pair of oligonucleotides produces, after amplification of genomic DNA, a 333 bp fragment, in which a G / C substitution at position 10141 of AB020864 (in exon 13) (resulting in a mutation
Glu / Gln) generates a restriction site for the enzyme Hinfl.

- oligonucleotide B1310: 19-mer sense (SEQ ID N0: 73), located in the intron which precedes exon 13 (SEQ ID NO: 69): positions 10198-
10216 of AB020864 and - oligonucleotide B1311: 19-mer antisense (SEQ ID N0: 74), located in the intron following exon 13 (SEQ ID NO: 69): positions 9979-9997 of AB 020864.

 This pair of oligonucleotides produced after amplification of genomic DNA, a 238 bp fragment, in which a G / C substitution at position 10141 of AB020864 (in exon 13) (causing a Glu / Gln mutation) generates a site restriction for the enzyme Hinfl: three fragments of 132.71 and 35 bp are obtained when the DNA is mutated and two fragments of 203 and 35 bp when the DNA is wild-type. c) amplification of exon 1 in order to detect the C / T substitution (Gin / Stop): - oligonucleotide B1314: 22-mer sense (SEQ ID NO: 31), located in exon 1: positions 10977-10998 from AB020865; and - oligonucleotide B1315: 22-antisense (SEQ ID NO: 32), located in exon 2: positions 566-587 of AB020865.

 This pair of oligonucleotides produces, after amplification of the mRNA (RT-PCR), a fragment of 516 bp (SEQ ID NO: 33), which should be sequenced, to detect the substitution C / T in position 10664 of AB020865 [creation of a stop codon in position 630 of hATIP3 (SEQ ID NO: 4)] and the substitution A / G in position 10987 of AB020865 [creation of a replacement of a His residue in Arg in position 522 of hATIP3 (SEQ ID NO: 4)].

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 4. fragments useful for detecting alternative splicing a) amplification of exon 3 with a view to detecting alternative splicing, making it possible to distinguish hATIP2 from hATIP3: - oligonucleotide B1312: 22-mer sense (SEQ ID NO: 34), loca - read in exon 2 (SEQ ID NO: 37): positions 562-583 of AB020865; and - oligonucleotide B1313: 22-antisense (SEQ ID NO: 35), located in exon 5 (SEQ ID NO: 41): positions 72687-72708.

 This pair of oligonucleotides produces, after amplification of the mRNA (RT-PCR), a fragment of 414 bp if exon 3 is present and a fragment of 354 bp if exon 3 is absent.

 The experimental conditions for amplification by PCR are similar for all the pairs of oligonucleotides: number of cycles: 30; hybridization temperature between 50 ° C. and 55 ° C., in the presence of Taq DNA polymerase Proméga, according to the supplier's specifications.

 The different nucleic sequences (cDNA, genomic sequences or fragments of these sequences) can be obtained by known methods, for example by extension of a nucleic sequence coding for an hATIP protein according to the invention by PCR or reverse PCR or by screening for genomic DNA banks by hybridization with a homologous probe, or by total or partial chemical synthesis.

 These different nucleic acid sequences make it possible either to determine the expression profile of the hATIP mRNA and a possible alteration of this profile in a tissue or a biological sample, or to highlight the hATIP gene, allelic variants of this gene and a possible alteration of this hATIP gene (changes, deletions, point mutations) and consequently assess the existence of a cancerous state.

 The different nucleic sequences defined above constitute good diagnostic tools for the detection of cancer cells which have mutated, deleted or rearranged the tumor suppressor gene.

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 correspondent (ATIP gene) and / or prognosis for the detection of cancer cells by detection of an alteration of the sequences or of the expression of the products of this gene.

 The present invention also relates to a detection reagent, consisting of at least one nucleic acid sequence as defined above.

 The present invention also relates to the use of a nucleic sequence chosen from the group consisting of the nucleic detection reagent as defined above, the nucleic sequence SEQ ID NO: 53 coding for hATIPl and the fragments of minus 15 bp of said sequence SEQ ID NO: 53 for the evaluation, prognosis and / or detection of a precancerous or cancerous state.

 The different nucleic acid sequences, as defined above, can also be advantageously used to construct expression vectors of an hATIP protein, capable of being used to produce said proteins or in gene therapy.

 The present invention therefore also relates to vectors, characterized in that they contain a sequence coding for all or part of a human hATIP protein as defined above. In such vectors, the sequence encoding the hATIP protein is under the control of appropriate transcription and translation control elements.

 These vectors can be obtained and introduced into a host cell by known techniques.

 The invention also relates to a eukaryotic or prokaryotic host cell transformed with a vector as defined above.

 The present invention also relates to isolated and purified ATIP proteins, characterized in that they are coded by a cDNA sequence or a transcript or a fragment of genomic nucleic acid, as defined above.

 More specifically, they include at least one

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 fragment common to all hATIP proteins, which corresponds to the region of interaction with the AT2 receptor selected from the group consisting of the sequences SEQ ID NO: 11 and SEQ ID N0: 12, which proteins do not include the sequence SEQ ID NO : 13, specific for hATIP1, in their N-terminal portion.

 Said proteins represent new isoforms interacting with the AT2 receptor.

 Even more precisely: - the hATIP2 protein has the sequence SEQ ID NO: 2 - the hATIP3 protein has the sequence SEQ ID NO: 4 - the hATIP4 protein has the sequence SEQ ID NO: 6 and - the hATIP5 protein has the sequence SEQ ID NO: 8.

 These different isoforms correspond to alternative splices of the hATIP gene: - hATIPl, which is the subject of the aforementioned application PCT / FR99 / 01908, corresponds to the splicing product of exons 7 to 16; - hATIP2 corresponds to the splicing product of exons 1 to 16 with the exception of exons 4bis and 7; - hATIP3 corresponds to the splicing product of exons 1 to 16 with the exception of exons 3, 4bis and 7; - hATIP4 corresponds to a splicing product identical to that of hATIP3; however its N-terminal part is shorter due to the presence of a stop codon in exon 1; this stop codon is due to a C / T substitution at position 10664 of the sequence n AB020865.

 - hATIP5 corresponds to the splicing product of exons 4 bis to 16, with the exception of exon 7.

 Advantageously, said hATIPs comprise particular motifs, located in the sequence SEQ ID NO: 10, common to all the ATIP proteins and containing the sequence SEQ ID NO: 11, corresponding to the region of interaction with the AT2 receptor; said grounds participate in

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the specific action of said proteins (see FIG. 2): - leucine closure motifs (leucine zipper): L (6X) L (6X)
L (6X), in positions 214-235 and in position 266-287, with reference to the sequence
SEQ ID NO: 10.

- coiled-coil domains, in positions 60-116 and in positions 140-
331, with reference to the sequence SEQ ID NO: 10.

 The coiled-coil domains favor protein / protein interactions and dimerization. The presence of a large coiled-coil domain in the sequences common to all hATIPs indicates that these hATIP proteins are capable of homo-or hetero-dimerization, and that it is this property which could ensure their interaction function. with the AT2 receptor, and possibly their role as tumor suppressors.

 The inventors have found that in certain tumors, mutations are observed at the level of said region of interaction with the AT2 receptor; we can find, for example, the E / Q mutation (position 231 of the sequence SEQ ID NO: 10); this mutation is located in the middle of the first leucine closure motif and in the middle of the coiled-coil region.

 Other motifs are located in specific parts of the various hATIP proteins (see FIG. 1):. HATIPl protein: 2 consensus N-myristylation sites, which correspond respectively to positions 20-25 and 30-35 of the sequence SEQ ID NO: 13; this signal allows a possible post-translational modification of the protein (myristylation) which targets the protein at the membrane. Such a signal could allow the interaction of ATIP with membrane proteins.

 . HATIP2 protein: nuclear localization [Nuclear Localization Signal (NLS)] bipartite, which corresponds to positions 10-25 of the sequence SEQ ID NO: 2; this signal potentially targets hATIP2 in the nucleus, and could allow the dimerization of ATIP with nuclear proteins.

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 . HATIP3 protein: 7 consensus N-myristylation sites (see Figure 1).

 . HATIP4 protein: 2 consensus N-myristylation sites.

 . HATIP5 protein: 2 consensus N-myristylation sites.

 These different proteins as well as the hATIPl protein have an action on the proliferation of cancer cells and find application in particular for the preparation of anticancer drugs; for example, re-introduction into cancer cells of the hATIP gene or of appropriate splicing products of the hATIP gene makes it possible to restore normal proliferation of tumor cells. All or part of said proteins can also be directly used in the treatment of tumors.

 The present invention also relates to an anti-tumor medicament, characterized in that it includes a naked nucleic acid sequence, coding for all or part of an hATIP protein, as defined above or a vector such as defined above, or a host as defined above, or all or part of an hATIP protein as defined above.

 The proteins according to the invention also include any protein (natural, synthetic, semi-synthetic or recombinant) from any mammal; they generally include an interaction domain with the AT2 receptor which has the sequence SEQ ID NO: 12 and comprise or consist of: - either a functional ATIP isoform, - or a functionally modified mutant of an isoform ATIP (found in cancer cells, for example).

 The term “functional protein” is intended to mean a protein which exhibits normal biological activity (inhibition of cell proliferation and anti-oncogenic or tumor suppressor effect).

 The term “functionally modified mutant of a protein” is intended to mean a mutant which comprises one or more mutations which induce a substantial modification of its activity. Such mutants are in particular

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 those detected in cancer cells.

 The hATIP mutants can have an identity of less than 70% with one of the hATIP isoforms, as defined above; however, the identity of the unmodified sequences of these mutants, when aligned with the sequences of the corresponding hATIP isoforms remains high and may be greater than 70%.

 The present invention also relates to fragments of the proteins as defined above, comprising at least 12 amino acids and in particular the fragments of sequence SEQ ID NO: 10 and 12 to 17.

 The present invention further relates to antibodies, characterized in that they are directed against an ATIP protein or a fragment of hATIP protein according to the invention.

 Antibodies directed against a functional or non-functional hATIP isoform or against the zone common to all of the hATIP domain isoforms can thus be obtained. They can advantageously be either polyclonal antibodies or monoclonal antibodies.

 Such antibodies find application as a diagnostic tool for the detection of cancer cells which have mutated or deleted the corresponding tumor suppressor gene (hATIP gene).

 The present invention also relates to a detection reagent, characterized in that it is selected from the group consisting of at least one protein as defined above, at least one protein fragment as defined above and at minus one antibody as defined above.

 The present invention also relates to the use of a protein sequence chosen from the group consisting of at least one protein as defined above, at least one protein fragment as defined above or at least one antibody such as defined above, the sequence SEQ ID NO: 54, the fragments of at least 12 amino acids of said sequence SEQ ID NO: 54 and the antibodies directed against said protein of sequence SEQ ID NO: 54 or its fragments for l , prognosis and / or detection

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 a precancerous or cancerous state.

 The present invention also relates to means for detecting and correcting precancerous or cancerous states.

 More specifically, the subject of the invention is methods which use the nucleic acid sequences and / or the protein sequences as defined above for the evaluation, diagnosis or prognosis of a precancerous state. or cancerous.

 For example, the subject of the invention is methods of diagnosing a cancerous or precancerous state in humans, which comprise: either the detection of the expression of a functionally modified mutant of the hATIP gene, in a nucleic acid sample, - either the detection of an alternative splicing (establishment of an expression profile of the different isoforms of hATIP, at the level of the mRNA or of the proteins), - or the detection of a loss of expression of one or other of the different isoforms of HATIP in terms of mRNA or proteins.

 The nucleic acid sequences according to the invention can advantageously be used in these methods.

 The present invention therefore also relates to a method for detecting a deletion, an insertion or a substitution in the hATIP gene, for evaluating the risk of developing a cancerous state, which comprises: - obtaining a nucleic acid sample, and - detecting the presence in said nucleic acid sample of a nucleic acid sequence coding for an hATIP mutant, using at least one nucleic detection reagent, such as as defined above and / or of the nucleic sequence SEQ ID NO: 53 coding for hATIPl and fragments of at least 15 bp of said sequence SEQ ID NO: 53.

 According to an advantageous mode of implementation of said method,

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 it includes for said detection:. bringing said nucleic acid sample into contact with a pair of primers capable of amplifying a region of said hATIP gene possibly containing a mutation and. detecting said mutation by sequencing the sequence obtained or by bringing said sequence into contact with at least one appropriate restriction enzyme and analysis of the fragments obtained with respect to a normal control sample.

 According to another mode of implementation of said method, it comprises, for said detection:. the isolation, from the genomic DNA of a patient, and the genomic DNA of a control subject whose hATIP gene contains no mutation, of a portion of the hATIP gene coding for an hATIP protein which includes either of the exons as defined above (SEQ ID NO: 36 to 52), and. hybridization of the genomic sequence of said patient and of said control subject with a labeled probe specific for said portion of sequence of the hATIP gene, then. direct detection of mismatches and / or contacting with an appropriate restriction enzyme and separation of the restriction fragments obtained.

 The present invention also relates to a method for detecting at least one isoform of hATIP, from a biological sample using polyclonal or monoclonal antibodies, as defined above and / or the antibodies directed against protein with sequence SEQ ID NO: 54 or fragments thereof of at least 12 amino acids.

 The present invention also relates to a method for establishing the expression profile of the hATIP mRNAs of a biological sample, characterized in that it comprises: - the taking of a sample

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- the extraction of mRNA, - either the reverse transcription (RT) of said mRNA and the successive or concomitant amplifications of the cDNAs obtained in the presence of primers specific for each isoform of hATIP, then the analysis of the products amplification obtained by electrophoresis, or the hybridization of said
MRNA with a specific probe, isolated from the nucleic sequences as defined above and - establishment of the hATIP transcription profile of said sample.

 The present invention also relates to a method for establishing the expression profile of the hATIP proteins of a biological sample, characterized in that it comprises: - the taking of a sample, - the bringing into contact of a cell lysate extracted from said sample with antibodies specific for the various isoforms of hATIP and - establishment of the expression profile of the hATIP proteins of said sample, by detection of the complexes formed.

 The invention also relates to kits or kits for implementing the various methods according to the invention.

 Said kits preferably comprise at least one pair of primers and at least one probe, optionally labeled, as defined above, and optionally appropriate restriction enzymes.

 They can be implemented in combination with amplification kits; they can also include negative and positive controls, molecular size markers for the electrophoresis gel, for example.

 Other kits according to the invention can include antibodies, optionally labeled, as well as reagents suitable for the detection of antigen-antibody complexes. ; may also include positive and negative controls.

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 The nucleic acids according to the invention can also be used for therapeutic purposes, for example to restore a tumor suppressor effect.

 In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the process which is the subject of the present invention, as well as to the accompanying drawings, in which: - Figure 1 illustrates a comparison of the amino acid sequences: hATIPl, hATIP2, hATIP3, hATIP4 and hATIP5, the consensus N-myristylation sites are underlined, - Figure 2 illustrates the amino acid sequence common to the different hATIP (SEQ ID NO: 10), in which the two leucine closure motifs are indicated in bold and the coiled-coil domains are underlined, - Figure 3 schematically illustrates the alternative use of different exons of the hATIP gene (chromosome 8p21 .-p22), allowing the production of 5 different forms of mRNA (hATIPl to 5): the location of the exons, relative to the Genbank sequences n AB020864 and n AB020865, is indicated by a double arrow che, the exons (3,4 bis and 7) used alternately are framed, the start of protein translation is represented by a flag, and the position of the stop codons is represented by a star, - Figure 4 illustrates the structure in introns and exons of the hATIP gene sequence: the location of the different exons is specified with respect to the Genbank sequences n AB020864 and n AB020865, - Figure 5 illustrates the analysis of the hATIP gene by Southern blot, on genomic DNA human from human tumor cells (lanes 1-3) or normal human placenta cells (lane 4), after cleavage with the restriction enzymes HindIII or EcoRI and hybridization with the probe (B1260, SEQ ID NO: 18), covering the exons 11 to 16 of the hATIP gene, FIG. 6 illustrates the analysis of the expression profile of the hATIP transcripts in human tumor cell lines of different

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origins: tracks 1 to 6: settler; lane 7: liver; lanes 8 and 9: lung; lane 10: breast; lanes 11: and 12: ovaries and lane 13: ENT sphere by Northern blot using the probe (B1260, SEQ ID NO: 18), covering exons 11 to 16 of the hATIP gene, - Figure 7 illustrates the detection , by PCR and restriction analysis, of the G / C substitution at position 10141 of the sequence AB020864 (exon
13), corresponding to the Glu / Gln mutation at amino acid 250 of SEQ ID
NO: 2, from human tumor cells: PCR products obtained from wild DNA before and after cleavage by the enzyme Hinfl (lanes 1 and
2), PCR products obtained from DNA mutated in position 10141, before and after cleavage by said enzyme (lanes 3 and 4), - Figure 8 illustrates the analysis of the expression profile of family proteins HATIP in human tumor cell lines of different origins: ENT sphere (track 1), prostate (tracks 2 and 11), colon (tracks 3, 6, 10 and 13), liver (track 4), lung (tracks 5, 8 and 12), ovary (lanes 7 and 9), by immunoblot using a rabbit polyclonal antibody directed against the sequence (SEQ ID NO: 10), common to the proteins hATIP.

 It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.

EXAMPLE 1 Detection of rearrangements of the hATIP gene in cancer cells by Southern blot, on genomic DNA, using the B1260 probe (SEQ ID NO: 18). a) Materials and methods
The extraction of total genomic DNA, the preparation and labeling of the probes as well as the hybridization by Southern-Blot are carried out according to the conventional techniques described in Sambrook et al., 1989. The samples of total genomic DNA, prepared from human tumor cell lines originating from tumors of different origins (breast, ENT sphere and ovary) or from normal cells derived from human placenta, are digested either

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with HindIII or EcoRI. The fragments obtained are separated on agarose gel and then transferred to a nylon membrane; the membrane is hybridized with the radiolabelled probe B1260 (SEQ ID NO: 18), in the buffer: 45% formamide, 0.2% BSA, 0.2% polyvinyl pyrrolidone, 0.2% Ficoll, 0.1% sodium pyrophosphate, 0.01% SDS, 0.05 mM Tris pH 7.5 and 0.9 M NaCl, at 45 C, washed at low stringency (2X SSC, 0.01% SDS at 45 C, then autoradiographed 72 h at -
80 C. b) Results
Analysis of the hATIP gene using the B1260 probe which covers exons 11 to 16 of said gene (FIG. 5), shows a different restriction profile in tumor cell lines of different origins (figure: breast ( lane 1), ENT sphere (lane 2) and ovaries (lane 3)), compared to the normal control cells (lane 4), arpes cleavage with the restriction enzymes HindIII and EcoRI.

EXAMPLE 2 Analysis of the expression profile of the transcripts of the hATIP gene in cell lines derived from human tumors of different origins a) Materials and methods
The extraction of the RNAs, the preparation and labeling of the probes as well as the hybridization by Northern blot are carried out according to the conventional techniques described in Sambrook et al., 1989. About 5 g of poly (A +) RNA prepared from human tumor cell lines originating from tumors of different origins (ENT sphere, tongue, ovary, lung, colon and liver) are separated on agarose gel and then transferred to nylon membrane; the membrane is hybridized with the radiolabelled probe B1260 (SEQ ID NO: 18), in the buffer: 45% formamide, 9% Dextran-sulfate, 0.2% BSA, 0.2% polyvinyl pyrrolidone, 0.2% Ficoll, 0.1% sodium pyrophosphate, 0.01% SDS, 0.05 mM Tris pH 7.5 and 0.9 M NaCl, at 45 C, washed at low stringency (2X SSC, 0.01% SDS at 45 C, then autoradiographed 72 h at -80 C. b) Results

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FIG. 6 shows, in human tumor cells derived from tumor of different origins (breast, ENT sphere, ovary, lung, colon and liver), the different levels of expression of transcripts of 7.2 kb; 5kb;
3.5kb and 2kb; the transcripts hATIP2, hATIP3 and hATIP4 are contained in the 7.2 kb hybridization product. The other hybridization products, which have different sizes, contain transcripts which have not yet been identified.

EXAMPLE 3 Comparative amplification of a 238 bp fragment: Detection of a mutation on genomic DNA, by PCR and restriction analysis. a) Materials and methods
The genomic DNA is extracted from the tumor cells as described in Example 1, the PCR amplification is carried out using DNA Taq polymerase (Proméga), according to the protocol recommended by the producer, in the following experimental conditions: cycles: 30; hybridization temperature: 55 C.

 The detection of the G / C substitution at position 10141 of the sequence AB020864 (exon 13) is carried out according to the following protocol: - comparative amplification of a 238 bp fragment of exon 13 using the primers SEQ ID NO : 73 and SEQ ID NO: 74, in a sample of tumor cells to be analyzed, and in a sample of normal cells (control).

- digestion with HinfI - separation of the products obtained in 2% agarose gel: the presence of two cleavage products (203 and 35 bp) is characteristic of a wild DNA sequence. The substitution G / C at position 10141 of the sequence AB020864 generates an additional restriction site for the enzyme Hinfl. The detection of three DNA fragments (132.71 and 35 bp) is characteristic of a mutated DNA sequence. b) Results
Figure 7 shows the detection of G / C substitution in po-

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 sition 10141 of the sequence AB020864 (exon 13), corresponding to the Glu / Gln mutation at amino acid 250 of SEQ ID NO: 2, from human tumor cells. The presence of two cleavage products (203 and 35 bp) is characteristic of a wild-type DNA sequence. The detection of three DNA fragments (132, 71 and 35 bp) is characteristic of a mutated DNA sequence.

 EXAMPLE 4: Production of antibodies.

1. Production of antibodies to all hATIP proteins
An anti-hATIP polyclonal serum is produced in rabbits by injection of a fusion protein comprising the sequence SEQ ID NO: 11 fused to 6 histidine residues.

 These polyclonal antibodies used in immunoblotting identify three major polypeptides of apparent molecular weight: 32kDa, 64 kDa and 180 kDa, in cell lysates from different human tumor lines (see Example 5).

 These antibodies also detect the presence of a polypeptide of apparent molecular weight of 57 kDa, in the lysate of certain tumor cells. The detection of ATIP proteins of outliers in tissue lysates is a diagnostic method for the screening of certain cancers.

 2. Production of antibodies specifically recognizing one or the other member of the hATIP family. antibodies specific for the polypeptides hATIP2, hATIP3, hATIP4 and hATIP5 can be obtained by injection into rabbits of a peptide of at least 12 amino acids, comprising all or part of the sequence SEQ ID NO: 14; . antibodies specific for the hATIPl polypeptide can be obtained by injection into rabbits of a peptide of at least 12 amino acids, comprising all or part of the sequence SEQ ID NO: 13; . antibodies specific for the hATIP 3 polypeptide can

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 be obtained by injection into rabbits of a peptide of at least 12 amino acids, comprising all or part of the sequence SEQ ID NO: 15.

 . antibodies specific for the hATIP4 polypeptide can be obtained by injection into rabbits of a peptide of at least 12 amino acids, comprising all or part of the sequence SEQ ID NO: 16; antibodies specific for the hATIP5 polypeptide can be obtained by injection into rabbits of a peptide of at least 12 amino acids, comprising all or part of the sequence SEQ ID NO: 17.

EXAMPLE 5 Detection of an alteration of the expression of the hATIP proteins in cancer cells by the immunoblot method, using specific antibodies. a) Materials and methods
The protein samples obtained after lysis of the cells are assayed, separated by gel electrophoresis of 10% polyacrylamide, in the presence of SDS, then the proteins are transferred onto a nitrocellulose membrane and revealed using an anti-hATIP polyclonal antibody. , according to example 4. b) Results
Analysis of the expression profile of hATIP proteins in human tumor lines of different origins, analyzed by immunoblot using a polyclonal anti-hATIP antibody (FIG. 8), shows: the presence of three major polypeptides of weight apparent molecular: 32 kDa, 64 kDa and 180 kDa, corresponding to the proteins hATIP. In addition, the presence of an apparent molecular weight polypeptide of 57 kDa, corresponding to an aberrant size hATIP protein, is also observed in certain tumor lines. The detection of aberrant-sized hATIP proteins in tissue lysates is a diagnostic method for the detection of certain cancers.

 As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and

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 which have just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the framework, or the scope, of the present invention.

Claims (27)

1) Isolated cDNA sequence, characterized in that it is selected from the group consisting of: (a) the nucleic acid sequences coding for an hATIP protein, comprising at least one fragment common to all the hATIP proteins of sequence SEQ ID NO: 11, which corresponds to the interaction region with the AT2 receptor, which nucleic acid sequence does not include the sequence SEQ ID NO: 55; excluding the GENBANK sequences n AB033114 and n AL096842; (b) allelic variants of the nucleic acid sequence as defined in (a); (c) sequences complementary to the nucleic acid sequences, as defined in (a) and in (b) and (d) homologous sequences originating from any mammal.  2) cDNA sequence according to claim 1, characterized in that it comprises a sequence selected from the group consisting of the sequence SEQ ID NO: 9, a sequence corresponding to positions 364-714 of SEQ ID NO: 9 and a sequence which hybridizes under stringent conditions with the sequence SEQ ID NO: 9 and its fragments.  3) Sequence according to claim 1 or claim 2, characterized in that said sequence has one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 or their complementary sequences.  4) Genomic nucleic acid segment corresponding to the hATIP gene coding for an hATIP protein, which sequence is characterized in that it is selected from the group consisting of: (a) a sequence which corresponds to the concatenation of positions 12 636 to 1 of the sequence Genbank n AB020865 and positions 100,000 to 688 of the sequence Genbank n AB020864, which sequence codes at least <Desc / Clms Page number 29> NO: 2, 4, 6, 8 and 54) and which comprises at least 17 coding exons, the reading of which is done on the complementary reverse strand of said sequences n AB020865 and n AB020864; excluding said sequences n AB020865 and n AB020864; (b) allelic variants of the nucleic acid sequence as defined in (a); (c) mutants of the nucleic acid sequence as defined in (a) or in (b) which comprise at least the substitution of a nucleotide at at least one of said exons; (d) sequences complementary to the nucleic acid sequences, as defined in (a) and in (b) and (e) homologous sequences originating from any mammal.  for the proteins hATIPl, hATIP2, hATIP3, hATIP4 and hATIP5 (SEQ ID  5) Fragment of the genomic sequence according to claim 4, characterized in that it is selected from the group consisting of the sequences SEQ ID NO: 36-52 and SEQ ID NO: 56-72.  6) Fragment of the genomic sequence according to claim 4 or of the cDNA according to any one of claims 1 to 3, characterized in that it comprises between 15 bp and 1000 bp.  7) Fragment according to claim 6, characterized in that it is selected from the following fragments: sequence SEQ ID NO: 18-35.  8) Detection reagent, characterized in that it consists of at least one nucleic acid sequence according to any one of claims 1 to 7.  9) Expression vector of an hATIP protein, characterized in that it contains a sequence coding for all or part of a human hATIP protein according to any one of claims 1 to 4.  10) A eukaryotic or prokaryotic host cell transformed by a vector according to claim 9. <Desc / Clms Page number 30>  11) Use of a nucleic sequence chosen from the group consisting of the nucleic detection reagent according to claim 8, the nucleic sequence SEQ ID NO: 53 coding for hATIPl and the fragments of at least 15 bp of said sequence SEQ ID NO : 53 for the evaluation, prognosis and / or detection of a precancerous or cancerous state.  12) Isolated and purified ATIP protein, characterized in that it is coded by a cDNA sequence or a transcript or a segment of genomic nucleic acid, according to any one of claims 1 to 7.  13) Isolated and purified hATIP protein according to claim 12, characterized in that it comprises at least one fragment common to all the hATIP proteins, which corresponds to the region of interaction with the AT2 receptor, which protein does not include the sequence SEQ ID NO: 13, specific for hATIP1, in its N-terminal portion.  14) Protein according to claim 12 or claim 13, characterized in that it is selected from the group consisting of the sequences SEQ ID NO: 2, 4, 6 and 8.  15) Fragment of a protein according to any one of claims 12 to 14, characterized in that it comprises at least 12 amino acids.  16) Protein fragment according to claim 15, characterized in that it is selected from the following sequences: SEQ ID NO: 10 and 12-17.  17) Antibody, characterized in that it is directed against an hATIP protein according to any one of claims 12 to 14 or a hATIP protein fragment according to any one of claims 15 and 16.  18) diagnostic reagent, characterized in that it is selected from the group consisting of at least one hATIP protein according to claims 12 to 14, at least one protein fragment according to claims 15 and 16 and at least one antibody according to claim 17. <Desc / Clms Page number 31>  19) Use of a protein sequence chosen from the group consisting of the sequences according to claim 18, the sequence SEQ ID NO: 54, the fragments of at least 12 amino acids of said sequence SEQ ID NO: 54 and the antibodies directed against said protein of sequence SEQ ID NO: 54 or its fragments for the evaluation, prognosis and / or detection of a precancerous or cancerous state.  20) Method for detecting a deletion, an insertion or a substitution in the hATIP gene, for evaluating the risk of developing a cancerous state, which comprises: - obtaining a nucleic acid sample, and - detecting the presence in said nucleic acid sample of a nucleic acid sequence coding for an hATIP mutant, using at least one reagent according to claim 8, and / or of the nucleic sequence SEQ ID NO: 53 coding for hATIPl and fragments of at least 15 bp of said sequence SEQ ID NO: 53.  21) Method according to claim 20, characterized in that it includes: (a) bringing said nucleic acid sample into contact with a pair of primers capable of amplifying a region of said hATIP gene optionally containing a mutation and (b ) detecting said mutation by sequencing the sequence obtained or by bringing said sequence obtained into contact with at least one appropriate restriction enzyme and analyzing the fragments obtained with respect to a normal control sample.  22) Method according to claim 20 or claim 21, characterized in that it comprises for said detection:. the isolation, from the genomic DNA of a patient, and the genomic DNA of a control subject whose hATIP gene contains no mutation, of a portion of the hATIP gene coding for an hATIP protein which includes either of the exons as defined above (SEQ ID <Desc / Clms Page number 32>  NO: 36 to 52), and. hybridization of the genomic sequence of said patient and of said control subject with a labeled probe specific for said portion of sequence of the hATIP gene, then. direct detection of mismatches and / or contacting with an appropriate restriction enzyme and separation of the restriction fragments obtained.  23) Method for detecting at least one isoform of hATIP from a biological sample using antibodies according to claim 17 and / or the antibodies directed against the protein of sequence SEQ ID NO: 54 or its fragments at least 12 amino acids.  24) Method for establishing the expression profile of the hATIP mRNA of a biological sample, characterized in that it comprises: - the taking of a sample - the extraction of the mRNA - or reverse transcription (RT ) of said mRNA and the concomitant successive amplifications of the cDNAs obtained in the presence of specific primers of each isoform of hATIP according to any one of claims 5 to 7 and the analysis of the amplification products obtained by electrophoresis and / or specific hybridization mRNA with a probe according to any one of claims 1 to 7 and - establishing the hATIP transcription profile of said sample.  25) Method for establishing the expression profile of the hATIP proteins of a biological sample, characterized in that it comprises: - taking a sample, - bringing a cell lysate extracted from said sample into contact with antibodies specific for the different isoforms of hATIP, such as <Desc / Clms Page number 33>  as defined in claim 17 and - establishing the expression profile of the hATIP proteins of said sample, by detection of the complexes formed.  26) Kits or kits for the implementation of the different methods according to any one of claims 20 to 25.  27) Anti-tumor drug, characterized in that it includes a naked sequence according to any one of claims 1 to 7 or included in a vector according to claim 9 or a host cell according to claim 10 or all or part of an hATIP protein according to claims 12 to 16.