WO1999005529A1

WO1999005529A1 - Method for detecting a protein of the tubulin tyrosination-detyrosination cycle and applications

Info

Publication number: WO1999005529A1
Application number: PCT/FR1998/001644
Authority: WO
Inventors: Didier Job; Laurence Lafanechere; Robert L. Margolis; Jürgen WEHLAND
Original assignee: Commissariat A L'energie Atomique
Priority date: 1997-07-28
Filing date: 1998-07-24
Publication date: 1999-02-04
Also published as: JP2001511526A; FR2766573B1; FR2766573A1; CA2299168A1

Abstract

The invention concerns a method for detection and/or prognosis of cancer by detecting a protein of the tubulin tyrosination-detyrosination cycle (absence of tubulin-tyrosin-ligase (TTL) and/or presence of tubulin Δ2) and a detection and/or prognosis kit (metastatic potentiality, invasive character, rate of tumour development) of a cancer. Said method consists in detecting, in a tissue sample or in a biological fluid, a protein of the tubulin tyrosination-detyrosination cycle, selected in the group consisting of tubulin-tyrosin-ligase and tubulin Δ2 and/or an antibody directed against one of these proteins.

Description

DETECTION OF A TUBULIN TYROSINATION-DETYROSINATION CYCLE PROTEIN AND ITS APPLICATIONS

The present invention relates to a method for screening and / or prognosis of cancer by detecting a protein of the tyrosination-detyrosination cycle of tubulin (absence of tubulin-tyrosine ligase (TTL) and / or presence of tubulin Δ2) as well as a screening and / or prognosis kit (metastatic potential, invasiveness, speed of tumor development) of cancer.

Microtubules form, with the filaments ^of actin and intermediate filaments, the cytoskeleton of eukaryotic cells. Microtubules are protein organelles composed of tubulin molecules, assembled into tubular structures of about 25 nm in diameter, up to several tens of micrometers in length.

Microtubules play multiple roles in the cell. They play a central role in the genesis and maintenance of the shape of cells. During the interphase, they organize the intracellular space and are responsible for the intracellular transport of organelles. They are essential for cell division; in fact, during mitosis, they reorganize to form the mitotic spindle, responsible for the distribution of chromosomes between the two daughter cells. In the nervous system, microtubules are also directly involved in neurite growth and axonal transport.

Finally, they play a central role in the motility of differentiated cells, such as, for example, sperm.

In cells, tubulin, formed of two subunits (α and β), exists in several isoforms. This diversity is generated at two levels: - the selective transcription of one or more genes coding for each subunit,

- a variety of post-translational modifications of tubulin, such as its acetylation (Piperno G. et al., J. Cell Biol., 1985, 101, 2085-2094), its phosphorylation (Gard D. et al., 1985, J. Cell Biol., 1985, 100, 764-774), its glutamyation (Eddé B. et al., Science, 1990, 247, 83-85), its polyglycylation (Redeker V. and al., Science, 1994. 266, 1688-1691) or its tyrosination (Barra HS. et al., 1988, 2, 133-143).

This last modification is known as the tyrosination-detyrosination cycle of tubulin and is illustrated in FIG. 1. In accordance with this cycle, * p + 9Xtubulin exists in a tyrosine form (tyrosine tubulin or tubulin-tyr) and in a detyrosine form (dety- rosine tubulin or tubulin-glu); the passage from one to the other of these forms brings into play two specific enzymes which act at the level of the carboxy-terminal end of the α subunit of tubulin: the tyrosine residue is cleaved from the main polypeptide chain by a tubulin-carboxypeptidase (TCP) or added by a specialized enzyme, tubulin-tyrosine-ligase (TTL) (see Figure 1).

This cycle, which is absolutely specific to tubulin, includes the synthesis of a peptide bond without the participation of ribosomes.

Another tubulin isoform, resistant to tyrosination, has also been demonstrated (Paturle-Lafanechère L. et al., J. Cell. Science, 1994, 107, 1529-1543 and Biochemistry, 1991, 30, 10523- 10528). It is a particular form of tubulin, representing approximately 35% of cerebral tubulin, which has been called tubulin Δ2. It includes two fewer amino acids (Glu ⁴⁵⁰ -Tyr ⁴⁵¹ ), compared to tubulin-tyr (see Figure 2). This modification of tubulin relates to the site of action of TTL, which explains why this form of tubulin cannot be tyrosinated (Rϋdiger M. et al., Eur. J. Biochem., 1994, 220, 309- 320). A study of the localization of tubulin Δ2 has been carried out (Paturle-Lafanechère et al., 1994, cited above); it has shown that tubulin Δ2 is present in neurons (Purkinje cells, granular neurons) but cannot be detected in glial cells. In non-neuronal cells, tubulin Δ2 is normally absent, with the exception of spermatozoa and hair cells, where the microtubular structures are stabilized.

The Applicant has surprisingly found that there is a selective suppression of tubulin-tyrosine ligase (TTL) in tumors, which is directly correlated with the appearance of tubulin Δ2. The generation of Δ2 tubulin, during tumor growth, associated with the suppression of TTL activity is patho- logical; Δ2 tubulin therefore constitutes a particularly interesting tumor marker.

Consequently, the Applicant has set itself the goal of developing a test for screening and / or prognosis of tumors on the basis of these properties (function of TTL tumor suppressor and appearance of tubulin Δ2 in cancer cells).

The subject of the present invention is a method for screening and / or prognosis of a tumor, characterized in that it comprises the detection, in a tissue sample or in a biological fluid, of a protein of the tyrosination-detyrosination cycle tubulin, selected from the group consisting of tubulin-tyrosine ligase, tubulin Δ2 and / or an antibody directed against one of these proteins.

According to an advantageous embodiment of said method, said detection comprises:

- treatment of the tissue sample to concentrate and / or purify and / or dissolve the Δ2 tubulin or the cytoplasmic TTL.

- bringing said tissue sample or said fluid into contact with at least one reagent selected from the group consisting of tubulin-tyrosine ligase, Δ2 tubulin, the immunoreactive peptides derived from said proteins and the antibodies directed against these proteins and - the revelation of the complex, possibly formed, by any appropriate means, such as EIA, RIA, immunofluorescence.

By tissue sample is meant, within the meaning of the present invention, both a tissue homogenate and a tissue cut; this term also includes dissociated tissues, treated so as to return the cells to culture. Advantageously, to detect the tubulin Δ2 possibly present in a tissue biopsy, it is possible and this, without limitation, to treat the tissue sample, according to one of the following protocols:

- perform tissue sections, adequately fixed, and immunodetect the protein of interest; - subjecting, after freezing and grinding said tissue, the powder obtained to a suitable extraction buffer;

RECTIFIED SHEET (RULE 91) - optionally purifying and transferring the protein of interest;

- dissociate the cells and put them back in culture, immunodetect the protein of interest in these cells, put back in culture.

All methods of immunodetection and measurement of the interactions between antibodies and antigens can be used: Application to the detection of tubulin Δ2:

* Fluorimetric methods:

. direct method: a specific anti-tubulin Δ2 antibody is labeled with a fluorescent compound and brought into contact with the tissue sample; . indirect method: the unlabeled anti-tubulin Δ2 antibody is applied to the tissue sample; a labeled antibody directed against the Ig of the species from which the specific antibody originates, reveals its fixation;

* Radioimmunoassays

The technique is based on the link between the labeled Δ2 tubulin or a specific peptide (reproducing the specific sequence of the Δ2 tubulin) and a standard antibody specific for this tubulin. The mixture of these two reagents leads to the formation of soluble immune complexes, dissociable by cold Δ2 tubulin (or a specific cold peptide), possibly present in the sample to be analyzed. This technique requires physical separation of the Δ2 tubulin or of the free and bound radioactive peptide.

* Enzyme immunoassays

The principle is the same as that of radioimmunoassays, except that the antigen or antibody is covalently linked to an enzyme instead of being labeled as a radioisotope. The enzyme, usually horseradish peroxidase, alkaline phosphatase or β-galactosidase, is visualized by color reaction with the substrate. Several technical methods can be used:

. a competition technique can be used to measure the Δ2 tubulin. It is the tubulin Δ2 or a specific peptide which is always fixed on the microplates. A mixture of tissue sample, optionally treated as specified above, and small amounts of anti-tubulin Δ2 antibody is added. The the quantity of antibody fixed on the microplate is all the lower the more the Δ2 tubulin molecules present in the sample to be analyzed have neutralized more antibody molecules;

. sandwich methods can also be used to assay Δ2 tubulin in tissue samples that may be treated: microplates are coated with anti-tubulin antibodies; the sample to be analyzed is added and then an anti-tubulin antibody, different from the antibody fixed on the microplates, coupled to an enzyme is applied and revealed by the addition of the substrate. * Immunotransfer or western blot This test is more specifically carried out on samples in which the protein to be detected is dissolved; it comprises a separation and a transfer of the proteins before contacting with the reagent; for example, the cell or tissue sample is prepared by lysing the cells or the tissue homogenate with an adequate buffer, in the presence of protease inhibitors to avoid any enzymatic degradation.

The electrophoresis is carried out in a polyacrylamide gel according to the Laemmli technique, then the proteins, including optionally the Δ2 tubulin, of the gel are transferred to a nitrocellulose filter (or any other suitable filter); the nitrocellulose filter is recovered and incubated with the anti-tubulin antibody Δ2, then the tubulin Δ2-anti-tubulin antibody Δ2 complex is revealed by an anti-immunoglobulin antibody (secondary antibody) labeled with peroxidase (or with any other enzyme suitable) or by a radioisotope.

Application to the detection of circulating anti-tubulin Δ2 antibodies: * Enzyme immunoassay

. the indirect method is suitable for assaying anti-tubulin Δ2 antibodies. A specific peptide of tubulin Δ2 or tubulin Δ2 itself, is fixed on a microplate. The serum to be assayed is first pre-incubated with a peptide specific for excess tubulin-tyr, so as to eliminate the non-specific anti-tubulin antibodies from tubulin Δ2. Then, the serum to be assayed is added to the microplate and the binding of anti-tubulin Δ2 antibodies is detected by the addition anti-IgG antibodies, coupled to an appropriate enzyme and quantitatively revealed on the spectrophotometer after addition of the enzyme substrate;

Application to the detection of tubulin-tyrosine-ligase:

All of the methods set out above can be used to measure the TTL.

In tumor cells, TTL (tumor suppressor) disappears (negative signal) while tubulin Δ2 can be simultaneously detected (- appearance of a positive signal).

Although the method may include only detecting TTL, it is more advantageous to detect tubulin Δ2; indeed, a positive signal is easier to interpret than a negative signal (disappearance of TTL) and presents an amplification phenomenon.

According to another advantageous embodiment of said method, it comprises: the treatment of the tissue sample to obtain the soluble phase possibly containing TTL (in the presence of a non-denaturing buffer),

- bringing said tissue sample into contact with the tubulin-tyrosine ligase substrate, namely tubulin-glu, in the presence of tyrosine, possibly radiolabelled and - detecting the tubulin-tyr produced, by any suitable means.

This detection can be carried out either by measuring the incorporation of the radiolabelled tyrosine, or by an immunoassay, of the type described above for Δ2 tubulin, in the presence of an antibody specific for tubulin-tyr. The present invention also relates to a kit or kit for screening and / or prognosis of a tumor, characterized in that it comprises, for the detection, in a tissue sample or in a biological fluid, of a protein the tyrosination-detyrosination cycle of tubulin, selected from the group consisting of tubulin-tyrosine ligase, tubulin Δ2 and / or of an antibody directed against one of these proteins, in addition to useful quantities of buffers suitable for the implementation of said screening: - at least appropriate doses of a reagent selected from the group consisting of tubulin-tyrosine ligase, tubulin Δ2, the immunoreactive peptides derived from said proteins and the antibodies directed against these proteins; and

- appropriate doses of a development reagent. The present invention also relates to a kit or kit for screening and / or prognosis of a tumor, characterized in that it comprises, for the detection of tubulin-tyrosine ligase in a tissue sample, in addition to useful quantities of appropriate buffers for the implementation of said screening:

at least appropriate doses of a reagent selected from the group consisting of tubulin-glu, tubulin-tyr, the immunoreactive peptides derived from said proteins, the antibodies directed against these proteins and the reagents for assaying tyrosine; and, if necessary,

- appropriate doses of a development reagent.

The present invention also relates to the use of the tubu- A Δ2 as a tumor marker.

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 method which is the subject of the present invention, with reference to the accompanying drawings, in which: FIG. 1 illustrates the tyrosination-detyrosination cycle of tubulin;

- Figure 2 illustrates the differences in C-terminal sequences of the different tubulin isoforms;

FIG. 3 illustrates the immunofluorescent labeling of NIH / 3T3 cells, before the subcloning of TTL ^' cells (without tubulin-tyrosine ligase activity): FIG. 3A illustrates the double labeling of the Tyr and Glu microtubules, in the presence of 'monoclonal antibodies against tubulin-tyr (TYR) or polyclonal antibodies against tubulin-glu (GLU); FIG. 3B illustrates the double labeling of the microtubules Tyr and Δ2, in the presence of antibodies directed against tubulin-tyr (TYR) or of antibodies directed against tubulin Δ2 (Δ2). Some of the cells stain weakly with anti-tubulin-tyr antibodies and strongly with either anti-tubulin-glu antibodies or anti-tubulin Δ2 antibodies as indicated by the arrows (20 μm scale);

FIGS. 4 and 5 illustrate the comparative analysis in immunofluorescence of subclones of TTL cells ^" and of NIH / 3T3 TTL cells ^" , transfected stably with the TTL cDNA, for their content in Δ2 tubulin, tubulin-glu and tubulin-tyr. FIG. 4 illustrates the results obtained with the TTL ^" subclone and FIG. 5 illustrates the results obtained with the TTL ^' cell lines transfected and expressing TTL, in a stable manner. These different cells are subjected to double labeling, ie with a monoclonal anti-tubulin-tyr antibody (TYR) and a polyclonal anti-tubulin-glu antibody (GLU), or with a monoclonal anti-tubulin-tyr antibody (TYR) and a polyclonal anti-tubulin Δ2 (Δ2) antibody ( 20 μm scale);

FIG. 6 illustrates the immunoblot analysis of the content of tubulin-tyr, tubulin-glu, tubulin-Δ2 (FIG. 6 A) and TTL (FIG. 6B) of NIH / 3T3 cells (3T3), of TTL cells ^" (TTL ^" ) and TTL cells ^" transfected with the TTL cDNA (TTL ⁺ ). The cell extracts from the various cell lines are prepared as described below in Example 1. Samples (5 μl) are analyzed by procedure immunoblot to detect tubulin-α, using a monoclonal anti-tubu- line-α antibody (DM1A: "Tub) (Amersham); tubulin-tyr, using a monoclonal antibody primary anti-tubulin-tyr (YL1 / 2: TYR); tubulin-glu, using a polyclonal antibody primary anti-tubulin-glu (L3: GLU) and tubulin Δ2, using a primary polyclonal anti-tubulin Δ2 antibody (L7: Δ2). The major 50 kDa band observed corresponds to tubulin. In FIG. 6B, samples (5 μl) of cellular extracts are analyzed using a anti-TTL monoclonal antibody (ID3: TTL). The major band obtained from 43 kDa is the TTL;

FIG. 7 illustrates an immunoblot analysis of the total tubulin-α and tubulin-tyr, tubulin-glu and tubulin Δ2 content, of nude mouse tumors and of various normal mouse tissues. FIG. 7A corresponds to control cell extracts and to tumor cell extracts. Samples (5 μl) are analyzed in western blot to detect tubulin-α, tubulin-tyr, tubulin-glu and tubulin Δ2, under the conditions specified above for FIG. 6. 4 series experiments are carried out; the results obtained for each series include a control track representing the cell extracts used for the injection and two other tracks which correspond to the samples of tumors obtained from two different mice (mouse 1 and mouse 2). In the first series of experiments, which uses TTL cells ^" to induce tumors, the first track (control 17B12) corresponds to an extract of TTL cells ^" , obtained before injection into two different mice. The second track (mouse 1 (17B12)) and the third track (mouse 2 (17B12)) of this series of experiments correspond to samples of tumors, induced in these two mice after injection of the cells. The other series of experiments use TTL ^' cells transfected with a plasmid expressing TTL. In each of these three other series of experiments, each of the two mice (mice 3 and 4; mice 5 and 6; mice 7 and 8) was injected with TTL ^" cells transfected with TTL cDNA. -different clones of transfected cell lines were more particularly tested (GT4 (mice 3 and 4), 22C5 (mice 5 and 6) and 17F 12 (mice 7 and 8)). Figure 7B: several tissue samples of mice (heart, brain, muscle, liver, kidney and spleen) are loaded onto an 8% SDS gel at rates equivalent to that of total tubulin-α. The separated proteins are subjected to analysis in Western Mot, using either d '' anti-tubulin-α antibody (DM1A: α Tub), i.e. using an anti-tubulin Δ2 antibody (L7: Δ2); - Figure 8 shows the different tubulin isoforms and the TTL levels in _cells. TTL ^"transfected with a cDNA TTL set in vitro or in situ growth (emergence of sarcomas). FIG. 8A illustrates the results obtained with cells transfected with TTL cDNA (clone 17F12, mice 7 and 8), cultured either in the presence (control) or in the absence ((-) G418) of geneticin for 6 weeks; tumors are generated by injecting cells from the same clone into two mice (mice 7 and mice 8). The samples (5 μl) are analyzed in western blot to detect tubulin-α, tubulin-tyr, tubulin-glu tubulin Δ2, under the conditions specified above for FIG. 6. FIG. 8B: the same samples are analyzed in Western blot to detect TTL, using an anti-TTL monoclonal antibody (ID3); - Figure 9 illustrates the immunoblot analysis of Δ2 tubulin from different human tumors. FIG. 9A: the cells of a human breast cancer cell line (Cal 51) are either grown in vitro (culture) or injected into nude mice to induce a tumor (tumor); the cells are extracted and 5 μl of samples are analyzed in western blot, to detect total tubulin-α, using a monoclonal anti-tubulin-α antibody (DM1A: α Tub) and tubulin Δ2, to using a primary polyclonal anti-tubulin Δ2 (L7: Δ2) antibody. Figure 9B: small biopsies of different human tumors placed in nude mice to induce tumor growth. Tumor samples are analyzed by western blot to detect total tubulin-α and tubulin Δ2, under the conditions specified above. Figure 9C: samples (15 μl each), consisting either of a pool of a hundred cytosols (left lane), or by cytosols of different human breast tumors (one sample per lane) are analyzed in western blot for detect the tubulin Δ2 under the conditions specified above. 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: Preparation of samples and reagents for the implementation of the method according to the invention * Preparation of anti-tubulin Δ2 antibodies.

The antibodies are obtained according to the procedure described by Gundersen et al. (Cell, 1984, 38, 779-789).

The sequence of the peptide used as immunogen is EGEEEGE (7 amino acid residues of the C-terminal end of the Δ2 tubulin). This peptide is conjugated to KLH and injected into rabbits.

The IgGs obtained are purified according to the method of McKinney et al. (J. Immunol. Meth., 1987, 96, 271-278) and their specificity with respect to the Δ2 tubulin is verified by western blot analysis.

The anti-tubulin Δ2 antibodies (L7 antibody) produced are detected by an ELISA (Gundersen et al., 1984, cited above), in which the titration microplates are coated with tubulin-Δ2. * Preparation of anti-tubulin tyr antibodies

The anti-tubulin-tyr monoclonal antibody YL1 / 2 specifically recognizes the carboxy-terminal residue of the α subunit of tyrosine tubulin (Wehland J. et al., J. Cell Biol., 1983, 97, 1467-1475 ); it is obtained according to the procedure described by Kilmartin J.V. et al., J. Cell Biol., 1982, 93, 576-582).

* Preparation of anti-tubulin-glu antibodies

The sequence of the peptide used as immunogen is GEEEGEE (7 amino acid residues of the C-terminal end of tubulin-glu).

This peptide is conjugated to KLH and injected into rabbits.

The specificity of the antibodies obtained is verified by ELISA in accordance with the method described by Gundersen et al. cited above.

The IgGs obtained are purified according to the method of McKinney et al. (J. Immunol. Meth., 1987, 96, 271-278) and their specificity with respect to tubulin-glu is verified by analysis in western blot.

* Preparation of anti-TTL antibodies

Anti-TTL antibodies are obtained according to the procedure described in Wehland et al., J. Cell Biol., 1987, 104, 1059-1067). * Preparation of tubulin Δ2, tubulin-glu and tubulin-tyr purified

The different tubulins are isolated and purified from beef brains, as described in Paturle et al., 1989, cited above and Paturle-Lafanechère et al., 1991, cited above. * Preparation of biological samples.

In general, tissue samples can be prepared, either in a denaturing buffer (containing SDS, for example), or in any other buffer, possibly described in the prior art, which makes it possible to isolate the soluble phase of cellular or tissue homogenates. The choice of tampon depends on the subsequent use of the tissue sample. - Tissue samples:

. tissue samples from normal mice.

They are dissected and stored in liquid nitrogen until needed. The samples (heart, brain, muscle, liver, kidney and spleen) are weighed and homogenized in 1% SDS (1 ml / g of tissue), brought to the boil for 5 minutes, then centrifuged (200,000 g, 15 minutes at 20 ° C).

After addition of a buffer called “deposition” buffer (consisting of 125 mM Tris-HCl, 2% SDS, 10% glycerol, 10% 2-mercaptoethanol, 10% bromophenol blue, pH 6.8), l ^The sample is brought to the boil again for 3 minutes, then stored at -80 ° C until use.

. tumor samples.

They are obtained by injection of human tumor cells into nude mice; the induced tumor is recovered at different stages of its evolution.

More specifically, a human tumor cell line, called Cal 51 and described in J. Gioanni et al., Brit. J. Cancer, 1990, 62, 8-13) is cultivated on a DMEM medium supplemented with 10% calf serum; the cells are then injected into nude mice as follows: the cells are suspended in a growth medium and

400 μl of each cell suspension (from 1 to 2.5 × 10 ⁶ cells) is injected subcutaneously into the scapular zone of the nude mouse, under light ether anesthesia; the tissues are frozen in. liquid nitrogen, then reduced to powder (Freezer ^® mill,

Spex, Bioblock).

1 ml of lysis buffer / g of cellular homogenate is added (lysis buffer: Pipes-KOH 80 mM, pH 6.8, 1 mM MgCl ₂ , Triton-X-100 0.5% (vol / vol), glycerol 10% (vol / vol), CaCl ₂ 5 mM + cocktail of protease inhibitors). The lysis is carried out for 10 min at 4 ° C. Then, add 1/20 of the same buffer without CaCl ₂ , but containing 200 mM EGTA (EGTA 10 mM final), so as to chelate the CaCl ₂ .

The sample is then centrifuged (200,000 g, 15 min, 4 ° C). The supernatant is added with a deposition buffer (as above), brought to the boil for 3 min and stored at -80 ° C. until use. Another cell line, namely cells derived from NIH / 3T3 cells (ATCC CRL 1658, mouse embryonic fibroblasts), obtained after multiple passages in culture, can be used to prepare tumor samples, under the conditions set out below in example 2.. human breast cancer samples.

The samples, collected in the surgical wards of hospitals, are immediately frozen in ^liquid nitrogen and then pulverized as specified above. The extraction buffer in this case consists of 100 mM Tris,

1 mM EDTA, 2.5 mM MgCl ₂ , 10% glycerol, 25 μg / ml leupeptin, 0.5 mM PMSF, 1 mM vanadate and 0.5 mM DTT at pH 7.4.

Cvtosolic extracts from cells in culture Recultivation of cells is not compulsory. Extracts can be produced on tissue homogenates as described above. However, the re-culture of the cells can allow, on the one hand, an amplification and, on the other hand, a subsequent sorting, by cloning for example, which can favor the detection. For example, in a tissue or in a mixed population of cells, it may be difficult to detect a decrease in TTL. If we subclone and amplify these cells without TTL (very reactive in immunofluorescence with the specific antibody of tubulin-glu and with the specific antibody of tubulin Δ2), then we can detect by western blot, in ELISA, or in activity test, the decrease or loss of TTL.

For example, to produce cytosolic extracts from cultured cells, the tissues are treated with collagenase II (200 IU / ml) for 15 h at 37 ° C in an incubator. The dissociated cells are then washed 3 times with a growth medium without serum and a last time with a growth medium containing serum; the cells are transferred into 75 cm ² flasks containing 20 ml of complete medium and readapted to cell culture. To make the cytosolic extract, the cells are trypsinized and washed twice with PB S.

50 μl of lysis buffer containing 5 mM CaCl ₂ are added to the cell pellet and the mixture is incubated for 10 min at 4 ° C., to depolymerize the microtubules. EGTA (10 mM, final concentration) is then added from a stock solution and the sample is centrifuged (200,000 g, 15 min, at 4 ° C). An electrophoresis buffer (identical to the aforementioned deposition buffer) is added to the supernatant and the mixture is brought to the boil (100 ° C.) for 3 min. These samples are stored at -80 ° C. Such samples are preferably used to carry out western blots (soluble protein).

The protein content of the different samples varies from 1 to 3 mg / ml.

* Cell transfection The pig's TTL cDNA is inserted into the eukaryotic expression vector pcDNA3 (Invitrogen), which contains a gene for resistance to neomycin. The plasmids containing the insert are transfected into TTL cells ^" , according to the procedure described in Chen et al. (Mol. Cell. Biol., 1987, 7, 2745-2752). Briefly, cells in exponential growth are trypsinized , seeded in petri dishes at a rate of 5.10 ⁵ cells / 10 cm and incubated overnight in 10 ml of growth medium. For the transfection, 25 μg of plasmid DNA is mixed with 0.5 ml of CaCl ₂ 0, 25 M, 0.5 ml of 2 x BBS (BES 50 mM, NaCl 280 mM, Na ₂ HPO ₄ 1.5 mM, pH 6.9) and the mixture is incubated for 15 min at room temperature. DNA is added dropwise to the cells in culture which are maintained for 20 hours at 35 ° C., under an atmosphere enriched in CO ₂ (3%). The medium is then removed, the cells rinsed twice with the medium. growth, reseeded and incubated for 24 hours at 37 ° C., under an atmosphere enriched in CO ₂ (6%). The cells are then diluted according to a 1/10 ratio and incubated for 24 hours, before the selection of stable transformants using geneticin (750 μg / ml).

After two weeks of selection, the isolated clones are selected using cloning rings and incubated separately in a growth medium comprising 750 μg / ml of geneticin for amplification and screening by immunofluorescence; * Antibody dilutions

^The monoclonal antibody anti-tubulin-tyr (YL1 / 2) is used at a dilution of 1/1 000 for immunofluorescence and western words. The secondary antibodies used with this antibody are rhodamine-conjugated anti-goat antibodies (1/250) for immunofluorescence or peroxidase-conjugated anti-mouse antibodies (1/5000) for western Words.

The purified polyclonal anti-tubulin-glu and anti-tubulin Δ2 antibodies are diluted 1/500 and 1/1000 respectively for immunofluorescence and 1/100000 and 1/200000 for Western Words. The secondary antibodies used with these antibodies are anti-rabbit antibodies conjugated to fluorescein (1/250) for immunofluorescence and anti-rabbit antibodies conjugated to peroxidase (1/5000) for Western Words.

For Western Words, total tubulin-α is detected using an anti-tubulin-α monoclonal antibody DM1 A (Amersham) diluted to 1/1000, which is detected using a secondary antibody anti-mouse conjugated to peroxidase (1 / 5,000).

The anti-TTL monoclonal antibody (ID3) is diluted to 1/1000 for its use in Western Words.

* In vivo experiments The cells transfected with the TTL cDNA (or with the plasmid alone) are subcloned in a growth medium containing 750 μg / ml of geneticin, then the various tubulins (tyr, glu and Δ2) are detected by immunofluorescence. The pure clones (containing 100% tyr cells or 100% glue cells) are selected and amplified. The cells in exponential growth are trypsinized and centrifuged (1250 rpm, 5 min, room temperature), the cells are resuspended in a growth medium and 400 μl of each cell suspension (from 1 to 2.5 × 10 ⁶ cells) are injected subcutaneously into the scapular region of nude mice under light ether anesthesia. For each different clone, two mice are simultaneously injected. The animals are observed daily and the day on which the tumor appears is noted. The animals are killed by cervical dislocation after a period of tumor growth, then the tumors are extracted, weighed and cut up and treated as specified above. Example 2: Study of TTL cells ^" : evidence of a correlation with the presence of Δ2 tubulin and a tumorigenic state. - Selection of TTL cells ^"

The abovementioned NIH / 3T3 cells, derived from the ATCC CRL 1658 line, are cultured on DME medium supplemented with 10% calf serum and 1% fetal calf serum.

The TTL ^" subclones are obtained by limiting dilution, after 3 growth cycles.

They are selected according to the uniformity of their phenotype, by double staining in immunofluorescence, using either anti-tubulin-glu antibodies or anti-tubulin Δ2 antibodies, in combination with an anti-tubulin-tyr anticoφs.

The TTL ^" subclones are those which do not produce tubulin-tyr.

In these cells, the Glu microtubules represent almost all of the cytoplasmic microtubules, instead of the small subpopulation usually observed in the different cell types. Tubulin-tyr is scarce and is diffuse in cytoplasmic microtubules.

In addition, these cells spontaneously have, in large quantities, microtubules formed from Δ2 tubulin. - Demonstration of the presence of tubulin-glu and tubulin Δ2

The microtubular networks of these inteφhase cells react substantially, with anti-tubulin-glu anticoφs (Figure 3A).

Double-labeling experiments comparing the distribution of microtubules containing tubulin-glu and microtubules containing tubulin-tyr show that the cells which react with anti-tubulin-glu antibodies do not react with anti-tubulin antibodies -tyr (Figure 3A, arrow). FIG. 3B illustrates the NIH / 3T3 cells whose microtubules contain Δ2 tubulin; this cell population is distinct from that containing tubulin-tyr (Figure 3B, arrow).

Among the cells which have a strong tubulin-glue signal, an abundant presence of tubulin Δ2 is observed in the microtubules (FIG. 4).

The results obtained in immunochemistry are correlated with the western blot analysis.

When the gels are loaded on the same protein base, the tubulin α is approximately equivalent in the NIH / 3T3 cells and in the tubulin-glu subclone (FIG. 6A).

However, these lines differ in the C-terminal sequence of tubulin α. The tubulin-glu subclone contains practically no tubulin-tyr, while it abundantly contains tubulin-glu and tubulin Δ2 (FIG. 6 A).

- Demonstration of the absence of TTL The cellular extracts obtained, as specified above, are brought into contact with anti-TTL antibodies, to determine whether the abundance of microtubules containing tubulin-glu is correlated with l absence of TTL activity.

After incubation of the anticoφs, no TTL is detected in the subclones containing tubulin-glu (FIG. 6B). Transfection assays of these TTL ^" cells with a plasmid comprising the TTL cDNA restores TTL expression and activity (Figure 6B) at apparently normal levels, resulting in the disappearance of tubulin-glu and tubulin Δ2 (Figure 6A).

This illustrates the correlation which exists between the absence of TTL and the presence of tubulin-glu and tubulin Δ2.

This effect of transfection with a plasmid containing the TTL cDNA is also illustrated by immunofluorescence: a TTL subclone ^" contains tubulin-tyr in abundance, very low levels of tubulin-glu (FIG. 5) and not Δ2 tubulin (no reaction with anti-tubulin Δ2 anticoφs, Figure 5). - Relationship between the absence of expression of TTL. the presence of tubulin Δ2 and the emergence of a tumor.

Both TTL cells ^"that TTL ^cells" containing the plasmid expressing cDNA TTL when injected into nude mice produces tumors.

In the case of tumors derived from TTL ^" cells containing the plasmid expressing TTL, it is observed, in western blot, that the cells contain tubulin-glu and tubulin Δ2 in abundance (FIG. 7A).

The result is completely reproducible: 2 mice injected with the same cell line give the same results.

This demonstrates the interest and specificity of the detection of tubulin Δ2, as a marker for the loss of TTL activity, linked to the emergence of tumor cells.

Indeed, the Western blot results of biopsies of normal tissues show that they contain tubulin-tyr and tubulin-glu, but never tubulin Δ2 except in the brain (Figure 7B).

The increased presence of tubulin-glu and tubulin Δ2 is due to the loss of TTL activity, during the growth of the tumor (western blot in the presence of anticoφs anti-TTL, FIG. 8B). As a result, the presence of Δ2 tubulin in a tissue is the direct result of the suppression of TTL activity; TTL can therefore be considered as a tumor suppressor. Example 3 Detection of Tubulin Δ2 in Cancer Cells

ELISA test Carried out on titration microplates comprising 96 wells.

All incubations are carried out at 37 ° C.

Each well comprises 0.5 μg of tubulin Δ2 in a carbonate / bicarbonate buffer (100 mM, pH 9.5).

The microplates are incubated for 2 hours then washed 3 times with 0.1% PBS / Tween buffer (PBST), the anti-tubulin Δ2 antibodies and frac- Tissue samples obtained from biopsies are mixed and preincubated for 45 min before 50 μl of the mixture is added / well.

The concentration of anti-tubulin Δ2 anticoφs is kept constant at 2.5 μg / well. These microplates are incubated for 1 h and washed 3 times with

PBST, before introducing 50 μl of anti-rabbit goat IgG conjugated to β-galactosidase (dilution 1: 2,000 in 1% PBST / BSA) in each well.

After 1 h of incubation, the plates are washed 3 times with PBST and 50 μl of substrate (O-nitrophenyl-β-D-galactopyranoside or ONPG 0.8 mg / ml; 2- mercaptoethanol 7 μl / ml, in PBS) are added per well.

After 30 min of incubation, the reaction is stopped by adding 50 μl of 0.5 M Na ₂ CO ₃ , pH 9.5. The absorbance is measured at 405 nm. Western blot

One can either extract the soluble fraction from the cells by any procedure of the prior art, or lyse the cells in a denaturing buffer as specified above.

The electrophoresis of the proteins obtained is carried out according to the method of Laemmli (Nature, 1970, 227, 680-685). The separated proteins are transferred to nitrocellulose membranes (Schleicher and Schuell, CeraLabo), according to the method of Towbin et al. (Proc. Natl. Acad. Sci. USA, 1979, 76, 4350-4344). The anti-tubulin Δ2 IgGs are diluted to 1/200 000.

The revelation is carried out by incubation of the membranes, for 30 min with secondary anticoφs conjugated to peroxidase, followed by 3 washes (Kit ECC Amersham). Immunofluorescence (IF)

- Sections:

Pieces of tumors are transferred just after surgical excision, in PBS containing 4% paraformaldehyde (pH 7.4), for 12 hours at 4 ° C. The fixed tissue is then incubated for 12 h in PBS containing 20% of sucrose and frozen for 1 min in isopentane, at -30 ° C. Sections (thickness 10 μm) are prepared using a cryotome at -18 ° C.

Immunofluorescence is carried out on floating sections, as described in Paturle-Lafanechère et al. (J. Cell. Sci., 1994). In routine, a double labeling is carried out, using simultaneously the anticoφs YL1 / 2, rat monoclonal specific for tubulin-tyr (Kilmartin JV et al., 1982, cited above), and the anticoφs L7, polyclonal produced in rabbit, specific for tubulin Δ2 (Gundersen et al., 1984, cited above). The quality of the detection of tubulin Δ2 has been greatly improved by the purification of the anticoφs L7 on the heptapeptide corresponding to the C-terminal end of tubulin Δ2.

We then proceed as described in Paturle-Lafanechère et al. (J. Cell. Science, 1994, supra).

- Cells:

For the IF analysis of the cells, the procedure is as follows: the cells are cultured for 1-4 days on glass coverslips, rinsed in PBS at 37 ° C., fixed for 6 min in anhydrous methanol at -20 ° C, then washed in PBS / Tween 20 at 0.1% (v / v). The cells are then incubated with suitable primary and secondary antibodies, diluted in PBS containing 0.1% BSA. The nuclei are stained with Hoechst 33258 at 1 μg / ml in PBS containing 0.1% BSA. The coverslips are mounted and observed under a microscope.

Results

The results illustrated below are those obtained in immunofluorescence and western blot; similar results are obtained in ELISA, when the procedure described above is followed.

- Transformed cell line (- ^ human tumor) Cal 51 in culture.

In culture, the cell line contains normal levels of tubulin-tyr and tubulin-glu; it does not contain Δ2 tubulin, as illustrated in western blot (Figure 9 A), IF or ELISA. - Nude mouse with cancer after injection of the Cal 51 cell line.

The production of tubulin Δ2 is observed when there is actually development of a tumor (FIG. 9A), this indicating the loss of TTL activity. - Various human tumors: human biopsies were analyzed, from tissues of various origins after passage through nude mice (Figure 9B).

The different tumors tested are as follows: synovial sarcoma, liposarcoma, osteosarcoma, hemangiopericytome; all have tubulin Δ2, with the exception of osteosarcoma. - Primary human breast tumors: a pool of 100 extracts of human biopsies and 7 individual extracts were analyzed; Δ2 tubulin is clearly detected (Figure 9C, left column), except in an individual extract.

It is thus clearly observed, in many tumors, cells, either in the form of islands, or isolated, rich in Δ2 tubulin and poor in tubulin-tyr.

Example 4 Absence of Detectable TTL in Cancer Cells

The disappearance of TTL constitutes a negative signal for screening for a tumor.

- Assay by ELISA. the cell extracts are produced as specified above.

The TTL is purified by immunoaffinity chromatography using a monoclonal antico ants specific for the ligase (anticoφs LA / C4) as described in Schroeder et al. (J. Cell Biol., 1985, 100, 276-281).

The enzyme is then eluted with 3M MgCl ₂ in 25 mM KXMES stabilization buffer, pH 6.8, 100 mM KCl, 2 mM MgCl ₂ , 1 mM EGTA, 0.5 mM DTT, 20% glycerol ( v / v) and dialyzed against the stabilization buffer. We then proceed as specified above.

- Measurement of enzyme activity

The enzymatic measurements are carried out in 50 μl of a buffer for measuring the TTL activity containing moφholinoethanesulfonic acid K ⁺

100 mM (MES-KOH) (pH 6.8), EGTA 1 mM, MgCl ₂ 25 mM, ATP 5 mM, KCl 100 mM, 100 μM tyrosine and tubulin-glu (maximum 4.5 mg / ml); tyrosine incoφoration is measured in the presence of ³ H-tyrosine (2 μCi / 50 μl).

As a variant, it is possible to assay the tyrosine tubulin formed by immunodetection (ELISA or immunofluorescence), under the conditions specified above. For example, an anticoφs is used which specifically recognizes the carboxy-terminal residue of the α subunit of tyrosinated tubulin (Wehland et al., J. Cell Biol., 1983, 97, 1467-1475) at a dilution of 1 / 1 OOOème for immunofluorescence or western words.

The secondary anticoφs used is an anti-rat goat Ig conjugated with rhodamine (1/250) for immunofluorescence or an anti-mouse goat IgG (or anti-rat) conjugated with peroxidase for western words (1 / 5,000).

For ELISA, the secondary anticoφs used is an anti-mouse or anti-rat goat IgG conjugated to β-galactosidase (1/2 00 in 1% PBST / SAB). EXAMPLE 5 Detection of Circulating Anti-Tubulin Δ2 Antibodies

By ELISA or by western blot, under the conditions set out above.

Example 6: In vivo study of the relationship between loss of TTL and metastases in animal models. Tumor lines are injected into the nude mouse. Tumor cells, originating from. the primary tumor and any metastases are then recovered, analyzed, readapted to in vitro culture and cloned.

Analysis shows that TTL cells ^" are much more abundant in metastases than in the primary tumor. After cloning of these cells, TTL cells ^" seem to have major adhesion defects. Restoration of a normal adhesion phenotype should be possible, by transferring these cells stably with the TTL cDNA.

As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all variants which may come to mind of the technician in the field, without departing from the scope or the scope of the present invention.

Claims

1 °) Method for screening and or prognosis of a tumor, characterized in that it comprises the detection, in a tissue sample or in a biological fluid, of a protein of the tyrosination-detyrosination cycle of tubulin, selected in the group consisting of tubulin-tyrosine ligase and tubulin Δ2 and / or an anticoφs directed against one of these proteins.

2 °) Method according to claim 1, characterized in that said detection comprises:

- treatment of the tissue sample to concentrate and or purify and / or solubilize the Δ2 tubulin or the cytoplasmic TTL;

- bringing said tissue sample or said biological fluid into contact with at least one reagent selected from the group consisting of tubulin-tyrosine ligase, Δ2 tubulin, immunoreactive peptides derived from said proteins and the anticoφs directed against these proteins; and - the revelation of the complex possibly formed by any suitable means.

3 °) Method according to claim 1, characterized in that it comprises:

- treatment of the tissue sample to obtain the soluble phase possibly containing TTL;

bringing said tissue sample into contact with the substrate of tubulin-tyrosine ligase, namely tubulin-glu, in the presence of tyrosine, possibly radiolabelled and

- the detection of the tubulin-tyr produced, by any appropriate means. 4) Kit or kit for screening and / or prognosis of a tumor, characterized in that it comprises, for the detection, in a tissue sample or in a biological fluid, of a protein of the tyrosination cycle- detyrosination of tubulin, selected from the group consisting of tubulin-tyrosine ligase and tubulin Δ2 and / or of an anticoφs directed against one of these proteins, in addition to the useful quantities of suitable buffers for the said screening: - at least appropriate doses of a reagent selected from the group consisting of tubulin-tyrosine ligase, tubulin Δ2, the immunoreactive peptides derived from said proteins and the anticoφs directed against these proteins; and

- appropriate doses ^of a staining reagent. 5) Set or kit for the detection and / or prognosis ^of a tumor, characterized in that it comprises, for the detection of tubulin-tyrosine ligase in a tissue sample, in addition to useful ^amounts' of suitable buffers for the implementation of said screening:

- at least appropriate doses of a reagent selected from the group consisting of tubulin-glu, tubulin-tyr, the immunoreactive peptides derived from said proteins, the anticoφs directed against these proteins and the reagents for assaying tyrosine; and, if necessary,

- appropriate doses of a development reagent.

6 °) Use of tubulin Δ2 as a tumor marker.