EP2066806A2 - Test pharmaco diagnostique ciblant l'oncologie - Google Patents
Test pharmaco diagnostique ciblant l'oncologieInfo
- Publication number
- EP2066806A2 EP2066806A2 EP07823487A EP07823487A EP2066806A2 EP 2066806 A2 EP2066806 A2 EP 2066806A2 EP 07823487 A EP07823487 A EP 07823487A EP 07823487 A EP07823487 A EP 07823487A EP 2066806 A2 EP2066806 A2 EP 2066806A2
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- EP
- European Patent Office
- Prior art keywords
- liv21
- protein
- cancer
- expression
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
Definitions
- the present invention relates to the field of medicine and biology. It concerns a new screening test and therapeutic monitoring in oncology. More particularly, it relates to diagnostic and / or therapeutic tests in oncology and neurodegenerative diseases.
- Molecular targeting by peptide and antibody vectors or siRNAs opens up a new understanding of the interdependence between diagnostic and therapeutic tools that now go hand in hand and open the way to pharmacodiagnostics.
- the understanding of the equilibrium between overexpression and under expression of genes according to their location in a cell compartment or another function of the proliferative state makes it possible to open perspectives of finer differential analysis.
- Age-related neurodegenerative diseases and cancers both involve a change in the physiological process of programmed cell death or apoptosis.
- Neuronal death is abnormally accelerated during neurodegenerative diseases such as Alzheimer's, Huntington's, Parkinson's etc.
- the process of cancerization corresponds to a blockage of apoptosis leading to an uncontrolled multiplication of cells.
- the link between these two processes has now become a major field of research in aging research. Control of the balance between cell division (mitosis), differentiation and programmed cell death (apoptosis) is fundamental during normal physiological processes, such as embryonic development, tissue regeneration and aging. Impairment of this balance can lead to major pathological conditions such as tumor formation or certain neurodegenerative diseases.
- Cancer is one of the leading causes of death worldwide. While, in the last generation, the percentages of disease-related deaths cardiac, cardiovascular and many other diseases are on the decline, the number of deaths related to different forms of cancer is increased. Despite the rapid advancement of our understanding of different forms of cancer, low survival rates are generally attributable to inadequate diagnosis and treatment. Most tumors can only be detected when they reach a size of about 1 cm. Since there is a relatively short period of time for the continuous development of a tumor to a stage that becomes incompatible with survival, this leaves little time for therapeutic intervention. So, early diagnosis becomes the key to success for cancer treatment.
- skin cancer is the most common cancer in Canada. In 1992 alone, 50,300 new cases of skin cancer were reported, compared to 19,300 cases of lung cancer, 16,200 cases of colorectal cancer and 15,700 cases of breast cancer. In other words, skin cancer is as common as the top 3 types of cancers combined. Its incidence continues to grow with its 64,200 new cases in 1997, an increase of 14,000 cases annually in 5 years. In particular, the incidence of malignant melanoma is growing at a rate of 2% per year. Early diagnosis remains the key to effective treatment. A malignant tumor is easily accessible and can be removed with minor surgery. In fact, the cure is 100% if skin cancer is detected early enough. However, early diagnosis of skin cancer remains difficult.
- Basal cell epithelioma or epithelioma squamous cell have a very different clinical development. Basal cell carcinoma spreads laterally over the surface of the skin without penetrating the deeper layers of the skin. Thus, although disfiguring, basal cell carcinoma rarely develops metastases and is rarely fatal. However, squamous cell carcinoma causes metastases and is often fatal. It is therefore important to be able to distinguish these two types of skin cancer and to be able to treat them locally by siRNA-type molecular targeting. A definitive diagnosis of skin cancer requires a biopsy and a histological analysis.
- Colon cancer is the third leading cause of cancer-related mortality among men and women in North America (16,200 cases per year). Early detection, leading to early intervention, has shown that treatment success and survival can be improved. For example, the 5-year survival rate is 92% for a patient whose disease has been detected at an early stage, while the rate drops to about 60% in patients with localized cancer, and to about 6% in those with metastases. However, only one-third of colon cancers are detected at an early stage. One of the reasons for this delay in diagnosis is the lack of a sensitive screening test, inexpensive and non-invasive.
- Breast cancer is one of the most common in women with colon cancer; The death rate is the highest of all cancers affecting women.
- E2F1, E2F2, E2F3, E2F4 etc. E2F-like transcription factors
- the p53 gene belonging to the family of tumor suppressor genes, blocks the cell cycle in the event of DNA damage. It has now been shown that this gene is also involved in the course of apoptosis (Oren 1994, Yonish-Rouach 1996).
- cyclin D1 one of the proteins constituting the regulatory subunits of the kinases of the cell cycle, essential for the progression of the cell cycle. This protein is also expressed during apoptosis in various cell types (Han et al, 1996, Pardo et al, 1996).
- the markers of the LIV21 complex will be the cytoplasmic markers at least equivalent and complementary.
- the present invention relates to a new cell cycle reinduction screening test targeting oncology.
- This is a diagnostic test and a prognostic test for different cancers (cancers of the breast, bladder, ovary, lung, skin, prostate, colon, liver, glioblastoma, sarcoma, leukemia, etc.).
- cancers cancers of the breast, bladder, ovary, lung, skin, prostate, colon, liver, glioblastoma, sarcoma, leukemia, etc.
- the invention relates to the use of genes or proteins of the LIV21 complex as well as their derivatives as therapeutic tools and as diagnostic and prognostic markers for cancers.
- the invention thus relates to the detection of.
- the invention lies in the manufacture of DNA, protein and antibody diagnostic microarrays comprising the known antibodies of the various proteins of the associated complex according to the phases of the LIV21 cell cycle, that is to say without restriction.
- Protein chips will make it possible to study protein interactions and post-translational modifications, more particularly phosphorylations and methylations. of certain proteins, which sign a characteristic state of the diseased cell different from the protein interactions and the metabolism of the healthy cell. The state of expression and silencing of certain genes being different.
- the chips with nucleotide sequences will make it possible to study in nuclear cell extracts and also cytoplasmic or membrane the sub expressions or the overexpressions of the genes and the ratio between genes and between proteins of the Liv21 complex and its associated partners.
- a first objective of the present invention is to demonstrate a method for detecting and prognosing cancer and its metastatic potential.
- the cancer is selected from cancers of the breast, bladder, ovary, lung, skin, prostate, colon, liver, sarcoma, leukemia and glioblastoma, without being limited to these.
- One aspect of the present invention is the use of the LIV21 complex as a prognostic indicator of cancer and in its therapeutic follow-up.
- the LIV21 complex is defined by the extract of proteins and peptides studied in mass spectrometry type Maldi and ESI MSMS or Maldi Tof Tof.
- the Liv21 complex is also defined by its overall mass spectrometry profile (FIG. 5) and the number and the molecular weight of the protein extract bands obtained on the acrylamide gels of FIGS. 1A and 1B, a function of the temperature at which the submitted sample and described migration conditions. Indeed, when, for example, the LIV21F peptide is located in the cytoplasm and is revealed directly by in situ hybridization, for example, or by biochip analysis, its highest expression in the cytoplasm, the cancer cells in the tissues are aggressive.
- the product of the expression of the LIV21 gene or the expression of a peptide such as LIV21F is preferentially localized in the cell nucleus, this is a prognostic indicator that the cells of the tissue are differentiated and quiescent and therefore non-invasive.
- the effectiveness of a cancer treatment can also be followed by the traceability of the LIV21 protein complex, its derivatives and the ratios with the associated proteins, but also by a diagmicroarray or biochip containing, among other things, this protein and its associated LIV21 complex ( Figure 20 and Figure 21).
- the detection of the protein kinase C epsilon (PKC ⁇ ) is also interesting since it has been determined that PKC ⁇ phosphorylates the LIV21 protein to maintain it in the cytoplasm. Thus, a significant increase in PKC ⁇ is indicative of the presence of cancer cells.
- the LIV21 / PKC ⁇ ratio increases in the cytoplasmic fraction of cancer cells. It is the same for the detection of HDACl which has been shown as LIV21 as involved in PML / SUMO / Rb / HDAC-1 complexes.
- the HDAC family overexpressed they cause the silencing of tumor suppressor genes, hence the interest of using HDAC inhibitors in therapy associated with other inhibitors regulating the metabolic cascade involving the LIV21 protein complex which contains the proteins LIV21, E2F4, Histones H4, H3, SUMO, PML, Rb, E2F1, etc.
- the detection of E2F1 and / or E2F4 proteins is of interest.
- the LIV21 protein forms a complex with E2F4 which is capable of inhibiting the expression of the E2F1 gene in the nucleus, the expression of the E2F1 gene being a sign of cell proliferation.
- a decrease in the association of LIV21 with the E2F4 protein is indicative of the presence of cancer cells.
- the presence of the E2F1 protein in the nucleus is indicative of the presence of cancer cells.
- the present invention relates to a method for the detection of cancer cells in a biological tissue sample (eg, breast, ovary, endometrium, bladder, melanoma, prostate, glioblastoma, etc.) of patients, which method comprises the detection of products of LIV21 complex gene expression in the nucleus compared to these same products in the cytoplasm of the cells in the tissue biological sample of said patient, a location of said LIV21 gene expression products in the cytoplasm being indicative of the presence of cancer cells and a location of said LIV21 gene expression products in the nucleus being indicative of the presence of non-cancerous cells.
- a location of some of the products of the expression of the gene complex into the cytoplasm LIV21 are indicative of the presence of invasive cancer cells and / or metastatic.
- the method according to the present invention further comprises the detection of the product of the expression of at least one gene selected from the group consisting of the protein kinase C epsilon (PKC ⁇ ) gene, the E2F1 gene and the E2F4 gene. .
- the method may include the detection of the product of the expression of two of these genes or three genes.
- at least one of the LIV21 / PKC ⁇ , LIV21 / E2F4 and LIV21 / E2F1 ratios can be determined in the present method. This ratio can be determined in the cytoplasm and in the nucleus separately preferably. Preferably, these ratios are determined in the nucleus. Preferably, these ratios are compared to those obtained in a healthy cell.
- the level of expression of each enzyme or polypeptide of the SUMO / Rb / HDAC complex or for certain cell types of the PML / SUMO / Rb / HDAC complex is an additional indicator of the proliferation state of the cell.
- the biological sample may be in particular a sample of blood, serum, saliva, tissue, tumor, bone marrow, circulating cells of the patient. This biological sample is available by any type of sampling known to those skilled in the art.
- the term "biological material” means any material making it possible to detect the expression of a target gene.
- the biological material may comprise, in particular, proteins, or nucleic acids such as in particular deoxyribonucleic acids (DNA) or ribonucleic acids (RNA).
- the nucleic acid may in particular be an RNA (ribonucleic acid).
- the biological material comprises nucleic acids, preferentially RNAs, and even more preferably total RNAs.
- Total RNAs include transfer RNAs, messenger RNAs (mRNAs), such as mRNAs transcribed from the target gene, but also transcribed from any other gene and ribosomal RNAs.
- This biological material comprises material specific for a target gene, such as, in particular, the transcribed mRNAs of the target gene or the proteins derived from these mRNAs, but may also comprise non-specific material of a target gene, such as in particular the mRNAs transcribed from the target gene.
- a gene other than the target gene, tRNAs, AKNr derived from other genes than the target gene and preferably the extracts to be studied will be analyzed when it comes to cell cultures directly on fresh crops with or without prior treatment aynt have undergone an extraction protocol separating the cell compartments.
- This type of kit known to those skilled in the art allows by a subtle buffer set to extract only the membranes or only that which is contained in the cytoplasm or the nucleus or the cytoskeleton and that in a differential manner.
- the Proteo extract kit (ref 539790) from calbiochem can be used for example.
- the other aspect of the present invention is the use of the genes and proteins mentioned above as markers of the invasiveness and metastatic aggressiveness of cancer cells of the prostate, colon, bladder, melanoma, ovary, endometrium, cervical cancer and cancers in neurology or ENT etc ..
- successive pharmaco diagnostic tests in the monitoring of a treatment will allow to observe by comparison -variations of gene expression levels or their silencing and thus to better evaluate the effectiveness of the treatments, readjust these treatments in the context of adapted combination therapy so that the physiological balance of the different products of the genes involved in these metabolic complexes are maintained.
- the gene expression product is detected at PAKNm.
- the mRNA can be detected by RT-PCR analysis (see examples attached). It can also be detected by. a Northern blot analysis.
- the gene expression product is detected at the level of the protein or peptides characterizing the LIV21 complex and its partners interaction.
- the protein and / or the associated LIV21 protein complex is detected with the aid of a specific antibody.
- the protein can be detected by Western blot analysis and SPR. In a preferred embodiment, it is detected by immunohistochemistry, immunocytochemistry, microfluidic, radiography or by peroxidase labeling.
- a significant increase in PKC ⁇ is indicative of the presence of cancer cells.
- the method may also include the determination of the LIV21 / PKC ⁇ ratio in the nucleus, the membranes and the cytoplasm. This ratio can be compared to that observed in a healthy cell. An increase in the LIV21 / PKC ⁇ ratio in the cytoplasmic fraction is indicative of cancer cells.
- the method comprises the detection of the combination of LIV21 with the E2F4 protein, and a decrease of this association in the cell nucleus being indicative. of the presence of cancer cells.
- the method may also include determining the LIV21 / E2F4 ratio in the nucleus and / or the cytoplasm. This ratio can be compared to that observed in a healthy cell.
- the method comprising detecting the expression product of the E2F1 gene
- the presence of the E2F1 protein in the nucleus is indicative of the presence of cancer cells.
- the method may also include determining the LIV21 / E2F1 ratio in the nucleus and / or the cytoplasm. This ratio can be compared to that observed in a healthy cell.
- the method comprises detecting a labeled RNA so as to target the specific sequence that characterizes it and thus signal the place of expression of the messenger RNA of the gene of interest.
- RNA gene E2F1 and E24 and PKC epsilon would allow a complementary diagnosis.
- the method according to the present invention allows in particular the detection of metastasized cancer, therapeutic monitoring and / or treatment recurrences.
- a second aspect of the invention relates to human LIV21 protein as well as fragments' thereof. More particularly, the present invention relates to an isolated, purified or recombinant human LIV21 protein comprising a peptide sequence selected from SEQ ID Nos. 1 to 5 or, more generally, from the peptide sequences characterizing it obtained by MALDI (FIGS. 3, 4 and 5). and NanoLC-ESI-MS.
- the polypeptide comprises the three peptide sequences SEQ ID Nos. 1 and 2 and 3.
- the LIV21 complex comprises proteins having a leucine zipper motif, a basic domain characteristic of the DNA binding domains. , and a nucleation sequence.
- the present invention relates to polypeptides whose sequence consists of a peptide sequence characterized by the spectrograms of FIGS. 3,4,5 of gel bands 1,2 and 3, selected from SEQ ID Nos. 1 and 2. and 3 and 4 and 5 and the hundreds of unordered additional sequences (see sequences listed in the appendix), the unmatched fragments identified in MALDI TOF analyzes (see FIGS. Unlabeled M (H +) masses partly characterize the LIV21 protein and some elements of its protein complex.
- a third aspect of the invention relates to an antibody that specifically binds to a polypeptide according to the present invention. More particularly, the antibody can bind specifically to a polype ⁇ tide comprising a peptide sequence selected from SEQ ID Nos 1-120 and preferably from SEQ ID No. 1 and 2 or 3 and / or 15 or 51 or a sequence having at least 80% identity with those ci.
- the present invention relates in particular to an anti-LIV21 serum manufactured by immunizing an animal or a human with a polypeptide according to the present invention, in particular a polypeptide comprising a peptide sequence selected from SEQ ID Nos. 1-120, preferably from the sequences herein and Or a sequence having 70.80 or 90% identity therewith.
- a fourth aspect of the invention relates to a kit for detecting cancer cells in a biological sample of a patient, the kit comprising one or more members selected from the group consisting of an antibody that specifically binds to human LIV21. according to the present invention and an anti-LIV21 serum according to the present invention, an oligonucleotide probe specific for the LIV21 mRNA and a primer pair specific for the LIV21 AKNm.
- the kit further comprises means for detecting the product of the expression of a gene or an oligonucleotide probe specific for the factor mRNA selected from the group consisting of the protein gene.
- epsilon kinase C PLC ⁇
- E2F1 gene the E2F4 gene.
- the antibodies as microarray RBP2 antibodies, SUMO 5 HDAC TNFalpha, CRB2, CycE / cdk2, cdkl,, CREBl and p300, Rb, pi 07, pl30, NFkB, cdc2A, mdm2, p21, p53, p65 and microarray with characteristic peptides known to those skilled in the art of the proteins mentioned above, their antigens being referenced. It is the combination of these different peptides corresponding to the specific interactions of the protein complexes which intervene in the dysregulation of the metabolism which generates the anarchic specific proliferation of the cancer or the neurodegeneration.
- the invention concerns the use of an antibody specific for human LIV21 cancer diagnostics and antibodies of the protein complex but specific antibodies RBP2, SUMO 5 FIDAC, TNFalpha, CRB2, CycE / cdk2, cdkl,, CREB1 and p300, Rb, p 107, p130, NFkB, cdc2A, mdm2, p21, p53, p65.
- the invention relates to the use of a specific primer pair or probe of LIV21 for the diagnosis of cancer.
- the diagnosis is made ex vivo on samples of a patient. (Puncture, biopsy, cell crushers, bronchial aspirations, DNA chips, Proteins, antibodies, hydrophobic support, or metal ions etc.)
- Figure 1 A one-dimensional gel (acrylamide gradient) and two-dimensional SDS Page characterizing the LIV21 complex.
- Figure IB g ⁇ l-bidffi ⁇ H ⁇ i ⁇ mel-andanalysis-of-ma-sses-e ⁇
- Figure 3A List of monoisotopic peaks in band 1 at 50 kD and band 2 at 49 to 50 KD
- Figure 3B is the profile of the protein LIV21 by mass spectrometry (Maldi) M
- Figure 4 is a profile of the spectrogram of band 1
- Figure 5 is the third spectrogram corresponding to the 12% one-dimensional 12% acrylamide gel band migrating at 51-52 KD and revealed by couch blue and the LIV21 antibody.
- FIG. 5bis is the list of the monoisotopic peaks of the third spectrogram corresponding to the 52 kD one-dimensional migrand acrylamide gel band and revealed by coupling blue and LIV21 antibody. Monoisotopic peaks with an M H + value. The masses are given with three digits behind the comma by the proteomic platforms because
- Figures 3-5 describe the MALDI assays giving a set of polypeptide sequences assignable to LFV21. and its complex.
- Example of chlatrine CLH22 The Mascot research parameters are: trypsin enzyme, variable modifications: carbamethylation and oxidation of methionines, no limit of molecular weight, without restriction of isoelectric point.
- Figure 7 same but second example: Mass type: monoisotopic. Mass error (MS): depending on the observer 50 ppm or 100 ppm.
- Non-tryptic cut 1 The masses seized are M
- Figure 10 PCR with primers showing a 1400bp band
- Figure 12 Gel 3 at 55 ° and molecular weight analysis
- Figure 13 Gel 4 at 45 ° and at 55 ° and molecular weight analysis
- Figure 15 1400 bp band ligation screen clones Cl to ClO
- Figure 16 Gel 5: screening ligation on the five new clones
- Figure 17 Gel 6 Screening of recombinant clones S55T and S55M and molecular weight analyzes.
- Figure 18 Examples of comparison of nucleotide sequences between the clones sequences.
- Figure 20 protein chip according to Yeretssian but with named peptides of the proteins of the complexes of interest studied in the invention.
- Figures 23 show by immunocytochemistry LIV21.
- the co-labeling (by fluorescent immunostaining) of LIV21 (green) and DNA (red to propidium) in the nuclei of the cells is red and here a variation of grayLIV21 (green) is cytoplasmic in colon cancer cells.
- FIG. 24 Peroxidase labeling of parallefied slides of normal breast tissue on healthy tissue biopsies and LIV21 nuclear expression in these biopsies
- the invention relates to the identification of antigens in cell lysates by immunoprecipitation.
- the analysis of the physical interaction of various associated proteins in the LIV21 complex such as E2F4 and E2F1 was studied by co-immunoprecipitation of protein complexes. This analysis revealed new markers with a diagnostic and prognostic utility for cancer (see PCT / FR2006 / 000510).
- Figure 1 and 2 From the one-dimensional and two-dimensional gel electrophoresis analysis ( Figure 1 and 2), the protein samples corresponding to the putative protein and the elements of the complex were extracted from the gels and digested with trypsin (Proméga) in order to to be analyzed by MALDI mass spectrometry ( Figure 3 to 5) and ESI MS / MS.
- Cloning has revealed about 20 clones out of 150 clones
- siKNAs were determined to allow for silencing-type regulation within this metabolic complex of interest for developing therapeutic applications (Figure 18).
- LIV21 In postmitotic cells, apoptosis could be an aborted attempt at mitosis. It is in this context that the application of LIV21 has been developed. The inventor has identified sequences of the LIV21 gene. Using LIV21 antibody on affinity columns he was able to extract peptides from the protein LIV21, he also used a second approach by coimmunoprecipitation kit (Pierce) to have larger-quantities of proteins (example 1) A From peptide sequences of the LIV21 protein obtained by mass spectrometry (Example 2), a primer design for amplifying a cDNA fragment was performed (Example 3).
- RNAs After culturing and amplification of MCF7 cell lines, extraction and purification of the RNAs, RT PCR and cloning in a shuttle vector were carried out, then screening of the resistant colonies and sequencing revealed sequences characterizing the genes of the LIV21 complex ( Examples 4 and 5). More than twenty characteristic clones on 150 clones were studied. . The cDNA of these clones was used to screen a library prepared from the total mRNA of MCF7 cells. The sequence of
- Deleted is correlated to nuclear for some, change role and function ,. On the other hand, they form the establishment of heterodimeric cell quiescen with other transcription factors, and some, bind to DNA.
- -j Deleted he
- the inventor then followed, using Northern blot, the expression of this new product during development, the embryonic stage. It has been observed that the amount of the LIV21 protein increases as the development progresses, that is, as the quiescent cell state is established. By the same strategy, he showed that the LIV21 / E2F4 complex was an inhibitor of E2F1 expression. This complex could correspond to a new checkpoint in the cell proliferation arrest.
- this new molecule LIV21 could have diagnostic value for the following reasons.
- the inventor was able to observe that, in all the proliferating tumor cells, this protein is cytoplasmic instead of being nuclear thanks to the LIV21 antibody ( example 8). It is therefore not in the good cell compartment to be active on stopping the multiplication of cells.
- LIV21 has thus been observed in mammals.
- the expression panel of LIV21 as a function of cell state was studied on tissues from different mammals. Protein analyzes on the different tissue samples confirmed that expression of this transcription factor appears to be associated with progression to a quiescent cell state (mitosis arrest and differentiation initiation).
- LIV21 is present in actively proliferating tumor cell lines and its expression is essentially cytoplasmic. The same results are obtained on human mammary adenocarcinoma (Example 9).
- the present invention relates to a new screening test for abnormalities of reinduction of the cell cycle.
- This pharmaco-diagnostic test is based on the study of the mechanism of action of the new gene, coding for a new potential transcription factor called LIV21, which negatively regulates proliferation.
- LIV21 is involved in stopping cell proliferation.
- the isolated cDNA fragment encodes a leucine zipper motif-containing protein, a basic domain characteristic of the DNA binding domains, as well as a nucleation sequence similar to the nuclear receptor binding factor OR1 1H6, cd53 antigen with the fragment of 400 bp.
- LIV21 which is cytoplasmic when the cells proliferate while it becomes nuclear when the cells become quiescent.
- this ubiquitous transcription factor has an expression and a localization regulated according to the cellular state: stronger expression and nuclear localization for the cells released from mitotic cycles, weak expression and cytoplasmic localization for actively proliferating cells such as human tumor cells.
- LIV21 appears as a key molecule to stabilize another transcription factor (E2F4) in the cell nucleus and thus induce cell proliferation arrest.
- E2F4 another transcription factor
- the present invention relates to the LIV21 complex and its nucleotide sequences which can also be used in the form of si RNA for therapeutic applications and also the LIV21 protein as well as derivatives and fragments thereof.
- the LTV21 protein is a protein which, according to the alternative splicing it undergoes, it presents itself in at least three different size forms ( Figure 7). Moreover, it can be phosphorylated or sumoylated. It has an apparent molecular weight between 50 kD and 51 kD in Western blot analyzes.
- It can be in the form of a dimer then having a molecular weight of more than 10OkD.
- LIV21a (SEQ ID No. 1) and LIV21b (SEQ ID No. 2) of SEQ N) I to 55 and the sequence
- EAPHQGPPQKPSQSAPGPTASAGSPPR (SEQ ID NO: 58)
- RRRPPPQRPHR (SEQ ID NO: 59)
- LAHGGARPHPCPDCPK (SEQ ID NO: 61)
- LAAHLWTHAPTRPYPCP (SEQ ID NO: 63) Homology with a functional segment of sulfotransferase 6 (OST6-Human /
- APMLLVALVLGAY (SEQ ID NO: 66)
- TLDGQITMEK (SEQ ID NO: 67)
- GEFLGQSEGVIEPNK (SEQ ID NO: 71)
- MAETVLRILDPVTCK (SEQ ID NO: 72) LLVASVGDDLQYHFER (SEQ ID NO: 73)
- NSQGVAWITLNSSIQK SEQ ID NO: 76
- VCTKPVESTIEDKIFGK SEQ ID NO: 77
- AGLQEEAQQLRDE (SEQ ID NO: 78)
- KAYECDITYGTNNEFGFDYLR (SEQ ID NO: 82)
- TMITEAGISKAEK (SEQ ID NO: 83) Homology with TCFL4 (17q21) in the basic region helix loop helix leucine zipper
- DLDQSV (SEQ ID NO: 112) EGF and HPV16
- ARWTFGGRDLPAEQPGSFLYDARL SEQ ID NO: 86
- Ferrodoxin NADP reductase Score at 60
- IPGGFFKR (SEQ ID NO: 87)
- VTDILKEGQEVEV (SEQ ID NO: 90)
- ADT_CHLKE ADP / ATP non-human translocase
- KTAVAPIERV (SEQ ID NO: 92)
- VAQQEGMKAFFK (SEQ ID NO: 97)
- FLDDLKTLDQK (SEQ ID NO: 103) Homology with BRCAl of the band at 50KD but at 100kd
- STR2 sequence selected from SEQ ID Nos: 1-110 or a sequence having 70%; 80% or, preferably, 90% homology therewith.
- the LIV21 complex has proteins comprising helical 3D structures which have a major functional role for its interactions with the rest of the LIV21 complex.
- the present invention relates to a purified human protein complex having a sequence comprising the sequence SEQ ID No. 1 and / or or SEQ ID No. 2 and / or SEQ ID No. 3 and / or SEQ ID No. 4 and / or SEQ ID No. 5.
- the LIV21F polypeptide comprises the sequences SEQ ID Nos. 1,2 and 5.
- the complex is studied with respect to a sequence selected from the peptide sequences characterizing it obtained by MALDI (FIGS. 3, 4 and 5).
- the invention also relates to the three peptides LIV21a (SEQ ID No.
- LIV21b SEQ ID No. 2
- LIV21e SEQ ID No. 5
- It also relates to peptides comprising at least 10 consecutive amino acids of human LIV21, preferably at least 20, 30 or 50 consecutive amino acids of the L1V21 peptides (see sequences 1- 120 in the appendix).
- the present invention also relates to a polynucleotide encoding the human protein LIV21, LIV21a and / or LIV21b, more generally a polynucleotide encoding a polypeptide according to the present invention.
- the polynucleotide encoding LIV21 may be RNA, cDNA or genomic DNA.
- the polynucleotides according to the present invention can be isolated from cells, more particularly from human cells, or from human cDNA libraries. They can also be obtained by a polymerase chain reaction (PCR) carried out on the total DNA of the cells or by RT-PCR performed on the total RNA of the cells or by chemical synthesis.
- PCR polymerase chain reaction
- the probes and primers described in the present application can be used to isolate and / or prepare a polynucleotide encoding a LIV21 complex protein. It further relates to a cloning vector or expression comprising such a polynucleotide.
- Such a vector may contain the elements necessary for the expression (expression vector) and possibly for the secretion of the protein in a host cell
- Said vectors preferably include: a promoter, translation initiation and termination signals, as well as appropriate transcriptional regulatory regions.
- the vector may be a plasmid, a cosmid, a BAC, a phage, a virus or other.
- the invention also relates to a transgenic non-human host cell or animal comprising a vector or polynucleotide according to the present invention.
- the invention further relates to derivatives of interest of LIV21 which are for example fusion proteins in which LIV21 is fused to marker proteins such as GFP.
- derivatives of interest of LIV21 which are for example fusion proteins in which LIV21 is fused to marker proteins such as GFP.
- the LIV21 complex proteins may be labeled by any means known to those skilled in the art.
- the present invention also relates to an antibody that specifically binds to a polypeptide according to the present invention, preferably human LIV21, a fragment or a derivative thereof.
- the antibody specifically binds to a LIV21a or LIV21b peptide.
- the antibodies may be polyclonal or monoclonal. It may also be fragments and derivatives of antibodies having substantially the same antigenic specificity, in particular antibody fragments (eg, Fab, Fab'2, CDRs), Humanized, polyfunctional, single-stranded (ScFv) antibodies, etc.
- the antibodies of the invention can be produced using conventional methods, including immunizing an animal and recovering its serum (polyclonal) or spleen cells (so as to produce hybridomas by fusion with lines appropriate cells).
- Said antibodies can be obtained directly from human serum or from serum of animals immunized with the proteins or peptides according to the present invention.
- Methods for producing polyclonal antibodies from a variety of animal species including rodents (mice, rats, etc.), primates, horses, pigs, sheep, rabbits, poultry, etc. are described for example in Vaitukaitis et al. (Vaitukaitis, Robbins et al., 1971).
- the antigen is combined with an adjuvant (eg, Freud's adjuvant) and administered to an animal, typically by subcutaneous injection. Repeated injections can be performed. Blood samples (immune serum) are collected and immunoglobulins are separated.
- the present invention relates to an anti-LIV21 serum manufactured by immunizing an animal with the polypeptide according to the present invention.
- the animal has been immunized with the peptide LIV21a and / or LIV21b.
- the animal is immunized with these two peptides.
- the peptides may be coupled to a carrier protein such as hemocyanin, and then injected into an animal, for example a rabbit, for immunization.
- Polyclonal antibodies were obtained from these two peptides by immunizing two rabbits and blowing a rabbit to have a preimmune serum.
- the subject of the invention is also the use of the antibodies according to the invention for the detection and / or the purification of the human L1V21 protein.
- antibodies specific for LIV21 can be used for the detection of these proteins in a biological sample. They thus constitute a means of immunocytochemical or immunohistochemical analysis or by microfluidics of the expression of LFV21 on sections of tissues.
- the antibodies used are labeled to be detectable.
- the antibodies can be labeled indirectly.
- the antibodies are labeled.
- Markers include radiolabels, enzymes, fluorescent, luminescent, chemical, magnetic particle markers, gold tagging, biotin / avidin labeling, peroxidase, etc.
- the subject of the invention is also a method for detecting the LIV21 protein in a biological sample, comprising a step of suitable treatment of the cells by any appropriate means making it possible to make the intracellular medium accessible, a step of bringing the said intracellular medium into contact as well as obtained with an antibody specific for the human LIV21 protein and a step of detection by any appropriate means of the LIV21 complex-formed antibody.
- the cytoplasmic and / or nuclear extracts are prepared, and these extracts are contacted with the human LIV21 protein specific antibody.
- the present invention teaches the development of the pharmaco diagnostic test. also allowing the follow-up of the evolution of a cellular proliferation.
- the present invention makes it possible to monitor the evolution of cell proliferation on fresh cells or tissues, on frozen cells or tissues and on tissues treated, inter alia, with paraffin.
- the applications can be the diagnosis of cancer as well as the follow-up of the evolution of a cellular proliferation.
- the cancer is selected from cancers of the breast, bladder, ovary, lung, skin, prostate, colon, liver, sarcoma, leukemia and glioblastoma, without being limited to these.
- transcription factor E2F4 to form a complex inhibiting the expression of factor E2F1 and the ability of LIV21 to translocate in the nucleus by specific inhibition of PKC ⁇ , sumoylation of LIV21 when it is nuclear and integrated into PML bodies and its interaction with HDAC.
- LIV21 different properties can be exploited by all means of optical imaging, sonic and spectroscopy current. Whereas the predominantly cytoplasmic state of this protein in cancer cases compared to its nuclear situation in healthy cells is a geographical and structural difference which makes it possible, without the need for a fluorescent marker, to differentiate spectral profiles of the functional pattern cancerous tissue versus healthy tissue and thus make the diagnosis.
- LIV21 and the nuclear localization of other proteins of this same complex is an indicator of the aggressiveness and the metastatic potential of cancer. Detecting the location of expression of the proteins' LIV21 of the complex indicates the presence of cells cancerous, more particularly invasive, aggressive and / or metastatic cancer cells.
- the invention further provides cancer diagnostic or prognostic methods for detecting the cytoplasmic localization of a nuclear localized transcription factor in healthy cells.
- the present invention relates to a method for detecting cancer cells in a biological sample of a patient comprising detecting the product of LFV21 gene expression in the nucleus and / or cytoplasm of cells in the biological sample of said patient, a location of said LIV21 gene expression product in the cytoplasm being indicative of the presence of cancer cells and a location of said product of LIV21 gene expression in the nucleus being indicative of the presence of non-cancerous cells.
- a location of said product of LIV21 gene expression in the cytoplasm is indicative of the presence of invasive and / or metastatic cancer cells.
- the method preferably comprises a prior step of suitably treating the cells contained in the sample by any appropriate means for making the intracellular medium accessible.
- the method optionally includes a step of comparing with a biological sample that does not contain cancer cells.
- the method according to the present invention further comprises the detection of the product of the expression of at least one gene selected from the group consisting of the protein kinase C epsilon (PKC ⁇ ) gene, the E2F1 gene and the E2F4 gene. .
- the method may include the detection of the product of the expression of two of these genes or three genes.
- at least one of the LIV21 / PKC ⁇ , LIV21 / E2F4 and LIV21 / E2F1 ratios can be determined in the present method. This ratio can be determined in the cytoplasm and / or in the nucleus. Preferably, these ratios are determined in the nucleus. Preferably, these ratios are compared to those obtained in a healthy cell.
- the gene expression product is detected at the level of PAKNm, PARNm can be detected by any means well known to those skilled in the art.
- the method according to the present invention also relates to the detection of a polynucleotide encoding the human LIV21 protein or a fragment thereof, for example LIV21a and / or LIV21b.
- the polynucleotide encoding LIV21 may be mRNA, cDNA or genomic DNA.
- the polynucleotides can be isolated from cells of the biological sample. They can also be obtained by a polymerase chain reaction (PCR) carried out on the total DNA of the cells or by RT-PCR carried out on the total RNAs of the cells or polyA RNAs.
- PCR polymerase chain reaction
- MRNA can be detected by RT-PCR analysis.
- the method uses a specific pair of primers of the expression product to be detected, in particular LIV21, PKC ⁇ , E2F1 or E2F4.
- specific primer pair is meant that at least one of the primers is specific for the expression product to be detected. That is, this pair of primers specifically amplifies a desired PARNm fragment.
- the RT-PCR analysis is carried out on nuclear and / or cytoplasmic extracts of the cells contained in the sample of the patient.
- the RT-PCR assay can be a quantitative analysis.
- a pair of specific LIV21 primers may be prepared based on the teachings of the present application.
- the primer pair may comprise the primers described in SEQ ID Nos. 3 and 4.
- MRNA can also be detected by NoRthern blot analysis.
- the method implements a. specific probe of the expression product to be detected, in particular LIV21, PKC ⁇ , E2F1 or E2F4.
- a probe specific to LIV21 can be prepared based on the teachings of the present application.
- An example of a specific probe comprises the sequence SEQ ID No. 5.
- the Northern blot analysis is performed on nuclear and / or cytoplasmic extracts of the cells contained in the sample of the patient.
- the nucleic probe is labeled.
- the technique of labeling oligonucleotides. is well known to those skilled in the art.
- the labeling of the probes according to the invention can be carried out by radioactive elements or by non-radioactive molecules.
- the non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, digoxygenin, haptens, dyes, luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent, phosphorescent agents.
- ligands such as biotin, avidin, streptavidin, digoxygenin, haptens, dyes, luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent, phosphorescent agents.
- luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent, phosphorescent agents.
- the probes specific for PKC ⁇ , E2F1 and E2F4 are well known to those skilled in the art.
- the gene expression product is detected at each protein of the Liv21 complex.
- each protein is detected using a specific antibody.
- the method comprises a step of contacting the cells of the biological sample with an antibody directed against each protein or peptide of the LIV21 complex.
- the antibodies can be monoclonal or polyclonal.
- the anti-LIV21F antibody may for example be an anti-LIV21F serum.
- the anti-Histone antibody can be a monoclonal antibody.
- the use of these antibodies as drugs that inhibit signal transduction should be done systematically after studying the expression profile of the cDNA or Antibody biochip or / and proteins.
- Chimeric antibodies are also part of this application (see, for example, the PML / RAR chromosome 15/17 fusion protein). It will be a function of the level of expression of the proteins or genes of each subject suffering from tumor, to adjust the dosage of the administration of one or more antibodies, siRNA and / or chemical treatments known to those skilled in the art and already used. in this type of pathology at its stage of evolution. It is at first akin to a pharmacogenomic treatment in the sense that it would be best adapted to the individual scale function of the pattern of expression of each gene and protein of interest and its expression rate in its cell compartment studied for each protein complex of interest .
- the method can use antibodies specific for the PKC ⁇ , E2F1 and E2F4 proteins, respectively.
- the polyclonal and monoclonal antibodies directed against PKC ⁇ , E2F1 and E2F4 are commercially available.
- PKC ⁇ of a polyclonal rabbit antibody (Santa Cruz Technology, sc-214), for E2F1, a polyclonal rabbit antibody (Santa Cruz Technology, sc-860), and for E2F4, a polyclonal rabbit antibody (Santa Cruz Technology, sc-866).
- the antibodies are labeled directly or via a secondary antibody. Antibody labeling techniques are well known to those skilled in the art.
- the protein can be detected by an immunoblot analysis (Western blot).
- Western blot analysis can be performed on nuclear and / or cytoplasmic extracts of the cells contained in the patient's sample. Briefly, the proteins are migrated in a gel and then transferred to a membrane. Then, this membrane is incubated in the presence of the antibodies and the Antibody binding is eventually revealed using labeled secondary antibodies.
- the protein is detected by immunohistochemistry, immunocytochemistry or immunoradiography. These techniques are well known to those skilled in the art. Immunocytochemistry analysis can be performed on whole cells from or derived from the sample, for example by cell culture. It can also be performed on isolated nuclei. Immunohistochemistry analysis can be done on sections of breast tissue.
- an immunocytochemical analysis may comprise the following steps. However, it is understood that other preparatory modes can be implemented. Cells from the biological sample are cultured, preferably on slides (Lab Tek, Nunc, Germany), then washed with buffer and fixed with paraformaldehyde (eg 4%). A saturation step is preferably performed by incubating the cells with S buffer (PBS-Triton X100 0.1% - 10% FCS). The cells are then incubated with the primary antibody and then washed and incubated with a fluorescent secondary antibody, if necessary. The nuclei can be labeled with propidium iodine (Sigma). The slides are moviol mounted for observation by fluorescence microscopy.
- isolated nuclei removed during nuclear extraction can be fixed with paraformaldehyde (eg 4%).
- the suspensions of nuclei are deposited between lamella and lamella and the observation is made by fluorescence microscopy and confocal microscopy.
- the primary antibodies are, for example, rabbit antibodies and the secondary antibodies are labeled antibodies directed against rabbit IgG.
- the biological samples come from a patient potentially suffering from cancer or cancer in a proven way.
- biological sample is meant in particular a sample of the biological fluid type, living tissue, tissue fragment, mucositis, organ or organ fragment, or any culture supernatant obtained using a sample.
- the method according to the present invention may comprise a step of taking a biological sample from the patient. The detection step can be performed directly on a section of tissue of the sample, on a culture of cells from the sample, on total cell extracts, nuclear and / or cytoplasmic extracts.
- a significant increase in PKC ⁇ is indicative of the presence of cancer cells. More specifically, the amount of PKC ⁇ in healthy cells is compared with the amount of PKC ⁇ in the cells of the sample and the significant increase is determined by this comparison.
- the method according to the present invention may optionally comprise the measurement of the LIV21 / PKC ⁇ rate. This LIV21 / PKC ⁇ ratio increases in the cytoplasmic fraction of cancer cells compared to healthy cells.
- the method comprises the detection of the combination of LIV21 with the E2F4 protein, and a decrease in this association being indicative of the presence of cancer cells. Detection of the association of LIV21 with the E2F4 protein can be achieved by concomitant detection of LIV21 and E2F4.
- the method according to the present invention may optionally comprise the measurement of the E2F4 / LIV21 level. This ratio E2F4 / LIV21 decreases in the nucleus of cancer cells compared to healthy cells.
- the presence of the E2F1 protein in the nucleus is indicative of the presence of cancer cells.
- the method according to the present invention may optionally comprise the measurement of the E2F1 / LIV21 level. This ratio E2F1 / LIV21 increases in the nuclear fraction of cancer cells compared to healthy cells.
- the method according to the present invention allows in particular the detection of metastasized cancer, therapeutic monitoring and / or treatment recurrences and to determine the degree of invasiveness of a cancer.
- detection based on LIV21 may be associated with the detection of other markers of cancer, in particular breast cancer, known to those skilled in the art.
- the present invention relates to a method of therapeutic follow-up of an anti-cancer treatment in a patient suffering from a cancer comprising administering the anti-cancer treatment to said patient and detecting cancer cells in a biological sample. of the patient according to the method of the present invention. A decrease in cancer cells will be indicative of the effectiveness of the treatment.
- the detection of cancer cells in a biological sample of the patient according to the method of the present invention may be carried out once or several times during the anti-cancer treatment or after the anticancer treatment.
- the biological sample is from the tissue affected by the treated cancer.
- the present invention also relates to a method for detecting recurrence following anti-cancer treatment of cancer in a patient comprising detecting cancer cells in a biological sample of the patient according to the method of the present invention.
- the detection of cancer cells in a biological sample of the patient according to the method of the present invention can be carried out once or several times after the anti-cancer treatment.
- the detection of cancer cells is indicative of recurrence.
- the biological sample is from the tissue affected by the treated cancer.
- the present invention also describes a kit for implementing a method according to the invention.
- the invention relates to a kit for the detection of cancer cells in a biological sample of a patient comprising one or more members selected from the group consisting of an antibody that specifically binds to human LF / 21 according to the the present invention and an anti-LIV21 serum according to the present invention, an oligonucleotide probe specific for LIV21 mRNA and a specific primer pair of LIV21 mRNA.
- the kit comprises antibodies that specifically bind to human LIV21.
- the kit comprises an oligonucleotide probe specific for the LIV21 mRNA. It may also include a probe specific for a house-keeping gene.
- the kit according to the present invention may comprise reagents allowing the detection of a LIV21 complex-an antibody produced during an immunological reaction.
- the kit according to the present invention further comprises means for detecting the product of the expression of at least one gene selected from the group consisting of the protein kinase C epsilon (PKC ⁇ ) gene, the E2F1 gene and the E2F4 gene.
- PDC ⁇ protein kinase C epsilon
- E2F1 gene the E2F4 gene.
- These detection means may be protein-specific antibodies, oligonucleotide probes specific for the mRNA concerned and / or a primer pair specific for the mRNA.
- the present invention also relates to a diagnostic composition
- a diagnostic composition comprising one or more elements selected from the group consisting of an antibody according to the present invention and a serum according to the present invention, an oligonucleotide probe specific for LIV21 mRNA and a pair of oligonucleotides. specific primers for L1V21 mRNA.
- LIV21 present in the nucleus.
- LIV21 being capable of self-nuclearization
- the production of an expression vector comprising a polynucleotide encoding human LIV21 could be considered for the purpose of overexpressing this protein in the nucleus of cells whose proliferation is to be regulated.
- the human LIV21 coding expression vector can be administered in vivo to the patient by any means known to those skilled in the art.
- the expression vector may be administered in the form of naked DNA (eg EP 465,529).
- naked DNA eg EP 465,529
- Microinjection techniques, electroporation, calcium phosphate precipitation, nanocapsule or liposome formulations are other available techniques.
- the expression vector may also be in the form of a recombinant virus comprising, inserted into its. genome, a polynucleotide encoding human LIV21.
- the viral vector may for example be chosen from an adeno virus, a retrovirus, in particular a lentivirus, as well as an adeno-associated virus (AAV), a herpes virus (HSV), a cytomegalovirus (CMV), a vaccinia virus, etc.
- AAV adeno-associated virus
- HSV herpes virus
- CMV cytomegalovirus
- vaccinia virus vaccinia virus
- the expression vector allows cell targeting.
- this vector could target cancer cells or the particular cell type that is affected by cancer. Targeting of a particular cell type can be achieved by placing the LIV21 coding polynucleotide under the control of a tissue-specific promoter.
- the expression vector may be targeted, for example by associating it with a specific molecule of a particular tissue or cancer cells, for example an antibody specific for an expressed molecule. specifically by the particular tissue or cancer cells.
- the choice of the expression vector can also influence the targeting. Indeed, if the expression vector is a virus, the tropism of the natural or modified virus may also allow some targeting.
- the present invention therefore relates to a pharmaceutical composition comprising a polynucleotide encoding LIV21, more particularly an expression vector coding for LIV21. It also relates to the use of a pharmaceutical composition comprising a polynucleotide encoding LIV21, in particular an expression vector coding for LIV21, as a drug. Preferably, the present invention relates to the use of a pharmaceutical composition comprising a polynucleotide encoding LIV21, in particular an expression vector encoding LIV21, for the preparation of a medicament for treating cancer.
- the present invention further relates to a method of treating cancer in a patient comprising administering to cancer cells a polynucleotide encoding LIV21, the expression of LIV21 to reduce or abolish the cancerous phenotype of the treated cells.
- the cancer is selected from breast, bladder, ovarian, lung, skin, prostate, colon, and glioblastoma cancers, without being limited thereto.
- PKC ⁇ in cancer cells it is possible to reduce the activity of PKC ⁇ in cancer cells.
- This decrease in activity can be achieved by decreasing the activity of the PKC ⁇ protein or by decreasing its expression.
- a decrease in the activity of the PKC ⁇ protein can be obtained by administering to the cancer cells inhibitors of the PKC ⁇ protein.
- Inhibitors of the PKC ⁇ protein are well known to those skilled in the art.
- a decrease in the expression of the PKC ⁇ protein can be obtained by using antisense or siRNA specific for the PKC ⁇ gene. Kits are commercially available.
- the techniques concerning inhibitions by antisense or siRNA are well known to those skilled in the art. (2004 Arya R, Lee W 2004, 2004 A Sen, Platet N 1998, Hughes 1987)
- the present invention therefore relates to a pharmaceutical composition comprising an inhibitor of the PKC ⁇ protein. It also relates to the use of a pharmaceutical composition comprising an inhibitor of the PKC ⁇ protein as a medicament, in particular for the preparation of a medicament for treating a cancer. Finally, it relates to a method of treating cancer in a patient comprising administering to the cancer cells an inhibitor of the PKC ⁇ protein, the inhibitor of the PKC ⁇ protein making it possible to reduce or abolish the cancerous phenotype of the treated cells.
- the inhibitor of the PKC ⁇ protein decreases the activity of the PKC ⁇ protein.
- the inhibitor of the PKC ⁇ protein decreases the expression of the PKC ⁇ protein.
- the cancer is selected from cancers of the breast, bladder, ovary, lung, skin, prostate, colon, liver, sarcoma, leukemia and glioblastoma, without being limited to these.
- PKC epsilon inhibitors are published and used commercially for other applications.
- LFV21 In the context of therapy of a neurodegenerative disease, it is possible to reduce the amount of LFV21 present in the nucleus. For this, one could reduce or block the expression of LIV21 in the nucleus of the cells affected by the neurodegenerative disease.
- the cells affected by the neurodegenerative disease are generally neurons, motor neurons, etc.
- the neurodegenerative disease is selected from Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS). .
- Inhibition or blocking of LFV21 expression can be achieved by any means known to those skilled in the art. In particular, by way of illustration, mention may be made of the antisense strategy, siRNA, and ribozymes.
- an antisense oligonucleotide or an expression vector encoding this antisense oligonucleotide could be prepared and used to block the translation of LFV21-encoding mRNA in vivo.
- a ribozyme can be prepared to cut and destroy in vivo the AKNm encoding LIV21. It is also possible to envisage a triple helix strategy in which an oligonucleotide is designed to hybridize with the gene encoding LIV21 and thus block the transcription of this gene.
- LIV21 nuclear localization of LIV21, for example by increasing the activity of PKC ⁇ in the cells affected by the neurodegenerative disease.
- Activation of PKC by DAG, PUFA oleic acid, linoleic acid, arachidonic acid, etc.
- PKC ⁇ protein Activation and Proteolysis of PKCs in Gonadotropic Cells: Macciano H Communication 2004, Junoy B, Mas JL Drouva SV UMR6544 Marseille).
- An increase in the expression of the PKC ⁇ protein can be obtained by using expression vectors coding for the PKC ⁇ protein and allowing it to be overexpressed in the cells affected by the neurodegenerative disease.
- the present invention relates to a pharmaceutical composition comprising an activator of the PKC ⁇ protein or an expression vector encoding the PKC ⁇ protein. It also relates to the use of an activator of the PKC ⁇ protein or of an expression vector encoding the PKC ⁇ protein for the preparation of a medicament for the treatment of a neurodegenerative disease.
- the invention relates to methods for the selection, identification, characterization or optimization of cell-depleting active compounds based on the measurement of the nuclear or cytoplasmic localization of LIV21, or the binding of LIV21 protein to the E2F4 protein.
- the selection, identification, characterization or optimization of active compounds of therapeutic interest comprises contacting a candidate compound with a cell and determining the nuclear or cytoplasmic localization of the cell.
- LIV21 expression product An increase in the nuclear localization of LIV21 indicates that the candidate compound is active to decrease or abolish cell proliferation.
- a decrease in the nuclear localization of LIV21 indicates that the candidate compound is active to treat or prevent a neurodegenerative disease.
- the selection, identification, characterization or optimization of active compounds of therapeutic interest comprises contacting a candidate compound with a cell and determining the level of expression of the gene. encoding the PKC ⁇ protein. A decrease in PKC ⁇ expression indicates that the candidate compound is active to decrease or abolish cell proliferation. An increase in PKC ⁇ expression indicates that the candidate compound is active to treat or prevent a neurodegenerative disease.
- the selection, identification, characterization or optimization of active compounds of therapeutic interest comprises contacting a candidate compound with a cell and determining the level of complex
- the selection, identification, characterization or optimization of active compounds of therapeutic interest comprises contacting a candidate compound with a cell and determining the level of expression of the gene. encoding the E2F1 protein. A decrease in E2F1 expression indicates that the candidate compound is active to decrease or abolish cell proliferation. An increase in E2F1 expression indicates that the candidate compound is active to treat or prevent a neurodegenerative disease.
- the subject of the invention is also a method for screening a compound capable of interacting in vitro, directly or indirectly, with LIV21, characterized in that: in a first step, the candidate compound is contacted with LIV21 and, in a second step is detected by any suitable means the complex formed between said candidate compound and LIV21.
- the subject of the present invention is also a method for screening a compound capable of modulating (activating or inhibiting) the activity of the LIV21 protein, characterized in that: in a first step, cells of a biological sample expressing the LIV21 protein with a candidate compound, in a second step, the effect of said candidate compound on the activity of said LIV21 protein is measured by any appropriate means, and in a third step candidate compounds capable of modulating said activity.
- the activity of LIV21 can for example be estimated through the evaluation of the ability of the cell to divide, by measuring the expression of the E2F1 gene, or by the cytoplasmic and / or nuclear localization of LIV21. .
- the candidate compound can be a protein, a peptide, a nucleic acid (DNA or RNA), a lipid, an organic or inorganic compound.
- the candidate compound could be an antibody, an antisense, a ribozyme or a siRNA.
- the cell line MCF-7 The cell line MCF-7
- the MCF-7 line is a non-clonal human breast adenocarcinoma cell line. During their differentiation induced by exogenous factors, these cells develop hypertrophy, membrane protrusions and a tendency to dissociate from each other. They acquire a secretory phenotype characterized by the appearance of numerous granules and secretory canaliculi.
- PLCs protein kinases C
- TNF for the induction of apoptosis
- TPA (12-O-tetradecanoyl phorbol-13-SUMOate)
- Mass spectrometry was performed for LIV21 protein and its complex.
- LIV21 protein was digested with trypsin.
- the peptides resulting from the digestion are solubilized in a solvent: acetonitrile / water (1/1) containing 0.1% TFA (trifluoroacetic acid).
- a saturated solution of the alpha cyano-4-hydroxycinnamic matrix was prepared in the same solvent. The same volume of the two solutions was taken, 1 ⁇ l was mixed and deposited on the MALDI plate for analysis.
- Mass spectrometry showed that the LIV21 protein and its complex digested with trypsin revealed a hundred peptides following the band of gel extracted between 49 and 54 KD and studied (see Figure 3 to 5).
- the LIV21 protein was characterized by a molecular weight of 50 kD, evidenced by Western Blot and two-dimensional SDS PAGE gel ( Figure 2). But there is a product of 10OkD at 130 kD which could be a dimer of LIV21.
- RNAs were extracted from two pools of 50 million cells with Nucleospin RNA L kit (Macherey Nagel) ref. 740,962.20 resulting in a pool 1 of 318 ⁇ g and a second pool of 182 ⁇ g.
- the poly A + RNAs were extracted from 313 ⁇ g of total pool 1 RNA using the kit oligo Tex m RNA Midi kit (Qiagen) ref. 70042.
- RNAs were retro-transcribed with the Revert Aid H minus M-MuLV Reverse Transcriptase Fermentas Ref. EP0451 lot 1124 with 3.64 ⁇ g of total RNA and 0.45 ⁇ g mRNA according to the supplier's conditions with an oligo dT primer. Reactions carried out at 2 different temperatures at 45 ° C. and 55 ° C. so as to eliminate the structures of the RNAs that can hinder the retrotranscription. H PCR
- PCRs were performed with the reverse transcripts as templates with primers Al + oligo dT at first. Nested PCRs were then performed on these first PCRs with primers Al + Splicing, Al + GDBRl, or ATG + Splicing, ATG + GDBR1.
- Enzyme Taq DNA polymerase polymerase Fermentas.
- Thermocycler Bio-Rad iCycler.
- the quality of the cDNAs was tested by amplification of household genes GAPDH, b-actin and Histone H3.3
- Figure 10 PCR with primers showing a band at 1400pb Figure 11 Gel 2 with molecular weight analysis Figure 12 Gel 3 at 55 ° and molecular weight analysis Figure 13 Gel 4 at 45 ° and 55 ° and molecular weight analysis Figure 14
- Ligation Screening Band 400pb Bl Clones at BlO Figure 15 Screening Ligation Band 1400 bp Clones Cl through ClO Figure 16 Gel 5: Ligation Screening on the Five New Clones Figure 17 Gel 6 Screening of Recombinant Clones S55T and S55M and Molecular Weight Assays.
- PCRs made from PCR matrices carried out with the Al + oligo dT primers on the RTs carried out at 45 ° C. on the total and poly A + (messenger) RNAs.
- the primers used for these PCRs are Al + GDBR1 or Al + Splicing reverse).
- Poly A RNAs were used to perform RT and are poorly observed at 1400 bp size (FIG. 10) for amplification with Al + GDBR1 primers and 415 bp with Al + Splicing reverse primers.
- Al + splicing there are other bands of 860 and 233 bp (FIG. 11).
- PCRs made from PCR matrices carried out with the Al + oligo dT primers on the RTs carried out at 45 ° C. on the total and poly A + (messenger) RNAs.
- the primers used for these PCRs are Al + GDBRl or Al + Splicing reverse).
- the primers used for these PCRs are ATG + GDBR1 or ATG + Splicing reverse).
- Figure 13 The nested PCRs performed with the ATG + GDBR1 primers give 1213 bp (RT 45 ° C) and 1559 + 1315 bp (RT at 55 ° C) bands, the expected theoretical size is 1455 bp.
- PCRs performed with the ATG + Splice reverse primers give more varied band profiles.
- the recombinant clones obtained were screened (after extraction of the plasmid DNA) by restriction with the Eco RI enzyme whose sites flank the insertion site of the PCR products into the pGEMT Easy vector.
- the different clones have a sequence of 450 bp in common
- the clone B2 the band which is about 400 bp is the same fragment as S55M1 which itself is the same fragment as S45T9 these fragments are between 400 and 450 bp approximately. This is not surprising because they were obtained after a nested PCR performed with the reverse splicing primer and the Al primers (for the B2 fragment) and the galgal ATG primer for the S45T9 and S55 Ml fragments. These fragments were obtained with RT made at different temperatures. On the other hand, the fragment S55T9 still has about 600 bp, one part of which (300 bp) has a rather strong identity with the other cloned fragments.
- EXAMPLE 6 From the cloning of the LIV21 gene described above, the new sequences are studied to draw the si RNA (see listing of si RNA and Figure 191 most specific and effective to create the "silencing" of the gene c ' that is to say, to inhibit its expression, knowing that the effectiveness of the inhibition at each injection of siRNA remains short, that is to say most often less than one hour. The inventor has developed diagnostic products and therapeutic products from the same tool that is siRNA.
- EXAMPLE 6.1 The inventor uses rhodamine or fluorescein labeled siRNAs or any other marker that can be revealed and monitored by optical observation, by a microscope in order to locate the place in the cells, tissues or labeled sample where will be directed if labeled RNA in order to cling to the specific sequence that characterizes it and thus signal the place of expression of PARN messenger of the gene of interest.
- the specific RNA can be used as a diagnostic marker as would be an antibody and allow to locate in a particular case as extemporaneous or any type of sample taken from a patient for example sample of cancerous tissue, fluorescence for any other labeling used on the si RNA and found in a cell compartment on the sample.
- EXAMPLE 6.2 for the therapeutic products, the inventor uses the if RNA of the complex LIV21 labeled in a first time to objectify their expected presence in the cell compartment and to visualize them then if RNA LIV21 unlabeled in a second time for their action purely therapeutic, in a particular case an injection of si RNA (fIg 19) in a neurodegenerative tissue or by an approach that allows if RNA to reach the tissue neurodegeneration (example of the ear, the eye , cerebrospinal fluid etc ..) and act by allowing proliferation to apoptosis and thus the death of the cell in neurodegeneration.
- EXAMPLE 7 Pharmaco diagnostic test
- the invention lies in the manufacture of DNA, protein and antibody diagnostic chips comprising the known antibodies of the various proteins of the associated complex according to the phases of the cell cycle at LIV21, that is to say the antibodies, peptides or nucleotide sequences of the genes: RJBP2, E2F4, E2F1, SUMO, INT2, CRB2, HDAC1, TGFbeta, integrin_alpha5 beta2, Myob, MyoD, cycE / cdk2, cdk1, chk1, chk2, TNFalpha, CREB1 and p300, Rb, plO7, p130 of the families pockets of protein.
- the protein chips ( Figure 20) will be able to study the overexpression or expression of gene products, protein interactions and post-translational modifications, especially the phosphorylations and methylations of certain proteins, which signify a characteristic state of the diseased cell. different from protein interactions and healthy cell metabolism. The state of expression and silencing of certain genes being different. This is the combined analysis of the results of overexpressions or subexpressions of the DNA microarray and the protein chip that will allow the best therapeutic targeting.
- EXAMPLE 7.1 Pharmaco-Diagnostic Chip (FIG. 20) Designed from Nucleotide or Peptide Sequences Fixed on Conventional Supports and According to the Known Agilent or Affymetrix or Caliper-type Techniques Without Restriction to Them and Corresponding to Known Gene and Protein Sequences following and listed elsewhere in the patent preferably using the sequences which in 3D analysis have a 3D structure preferably loop type or helix loop helix or basic loop or zinc finger or a 3D conformation similar to a corresponding helix most often at functional sites, or nucleotide sequences corresponding to a methylation zone of a cytosine, methylation of the promoter region of the gene leading to a silencing potential (see general bibliography) as well as to the novel sequences of LIV21 listed in the description and in annex but also common sequences to some onc and some viruses that are thought to be involved, particularly in breast cancer of Chinese populations: Mdm2 and HIVl: GA
- Example 7.2 Microfluidic test, for example Biacore type, using the SPR technique known to those skilled in the art based on a support fixed with a gold film allowing once the light beam sent towards the interface to obtain an energy absorbed depending on the presence and size of the protein complexes (if protein protein or antibody protein interaction) or protein complexes DNA (if DNA protein interaction) protein or peptide, and an evanescent wave perpendicular to the axis of the interface. (The inventor fixes the chosen peptide or DNA sequences on the gold particles and calculates for each interaction complex studied, the number of rUs depending on the size of the molecules mentioned in this patent.In microfluidic liquid passing on these chips.
- EXAMPLE 7 Study of the expression of LIV21 in biopsies d Breast cancer and colon cancer To determine whether the observations previously obtained are applicable to human tissues, a large number of cancer biopsies obtained from patients were studied by immunohistochemistry reaction with antibodies specific for the LIV21 protein complex. Immunohistochemical determination of LIV21 protein expression involved several patient biopsies. In addition, some paraffinic blades of breast cancer patients were also studied.
- FIGURE 23 Expression of the LIV21 protein determined by immunohistochemistry in colon cancer biopsies and healthy tissue biopsies. These results show that the cytoplasmic localization of LIV21 is an indicator of the aggressiveness - and the metastatic potential of cancer. The detection of LIV21 expression indicates the presence of invasive, aggressive and metastatic cancer cells. These results also show that the nuclear localization of LIV21 is an indicator of healthy quiescent cells of well differentiated tissues.
- PKCs Protein Kinase C
- TPA is a known activator of PKCs. It activates the growth of normal breast cells, does not alter the proliferation of benign tumor cells of the same tissue, but it drastically inhibits the proliferation of cells of human breast tumor lines such as the MCF-7 line. It reduces the cell growth of this line by positively controlling the c-erb-2 receptor and by negatively controlling the retinoic acid receptor, both of which are expressed particularly prominently in these cells.
- TPA strongly and rapidly inhibits estrogen receptor (ER) expression and function and induces the time-and dose-dependent translocation of cytosolic protein kinase C (PKCs) to membranes.
- ER estrogen receptor
- PKCs cytosolic protein kinase C
- TPA increases the capacity for migration of MCF-7 cells in vitro and a short treatment of these cells by TPA induces cell expansion and microtubule organization characteristic of their differentiation.
- the inventor verified the expression of LrV21 in these cells, at the transcriptional level and at the protein level.
- the inventor has discussed the study of the expression of the LFV21 protein by the Western blot technique, with an anti-LIV21 antibody, in the MCF-7 cells compared to the mammary tissues.
- Anti-LIV21 antibodies were obtained by the described method.
- LIV21 is expressed in mammary tissues as well as in MCF-7 cells, in the form of a doublet migrating at an apparent molecular weight of 50 kDa (
- the specific peptide sequences are sequences No. 1 and No. 2
- TPA induces proliferation arrest and differentiation of MCF-7 tumor cells. After 3 days of treatment, the control cultures present twice as many cells as the treated cultures. TPA at a concentration of 25 nM therefore inhibits proliferation.
- TPA-treated cells quickly acquire differentiated mammary gland cell characteristics: hypertrophy, membrane protuberances, and a tendency to dissociate from each other.
- the secretory phenotype (appearances of granules and secretory canaliculi) was not observed. .
- LIV21 in nuclear extracts was studied and performed after 12h, 24h, 48h, and 72h of treatment with TPA. During this kinetics, a maximum of anti-LIV21 immunoreactivity was observed from 12h, which is maintained until 48h and regains its initial intensity after 72h of treatment The immunoreactivity of LIV21 increases significantly at 12h, when the number of cells in S phase is minimal. It lasts until the recovery of the cell cycle observed at 72h.
- This example describes the conditions used for Western Blot analysis of breast cancer cells.
- the protein extracts are heated for 5 minutes at 80 ° C. in Laemmli buffer (pH 7.4, 0.06M Tris, 3% SDS, 10% glycerol, 1M PMSF, ⁇ -mercaptoethanol).
- Laemmli buffer pH 7.4, 0.06M Tris, 3% SDS, 10% glycerol, 1M PMSF, ⁇ -mercaptoethanol.
- SDS-PAGE sodium dodecyl sulfate-Poryacrylamide Gel Electrophoresis. 10 to 20 ⁇ g of proteins migrate in a 12% polyacrylamide gel for 1 h under denaturing conditions (Migration buffer: 25 mM Tris base, 192 mM glycine, 1% SDS, pH 8.3).
- the proteins are then transferred to the membrane of nitrocellulose (Schleicher & Schuell) for one hour in liquid transfer, in transfer buffer (25mM Tris, 192mM glycine, 20% methanol, pH 8.3).
- transfer buffer 25mM Tris, 192mM glycine, 20% methanol, pH 8.3.
- the membranes saturated in 0.1% PBS-Tween 0.1% -Triton 0.1% -5% skimmed milk for one hour, are contacted with the primary antibody diluted in PBS-0.1% Tween -Triton X100 0.1% -1% milk at room temperature with gentle stirring for one hour to two hours. After washing, the secondary antibody coupled to the peroxidase is incubated with the membranes.
- the revelation is by a chemiluminescence reaction using the ECL kit according to the supplier's protocol (Amersham).
- the primary antibodies used are:
- the anti-LIV21 serum which was manufactured using synthetic peptides based on the LIV21: PeptideLIV21a sequence (SEQ ID No. 1 and the LIV21b peptide (SEQ ID No. 2 and / or LIV21e peptide (SEQ ID NO: 51).
- the peptides were coupled to the hemocyanin before being injected into rabbits for immunization
- the polyclonal antibody was obtained from two of these peptides by immunizing two rabbits and blowing a rabbit to have a serum preimmun (to be sure that this antibody did not already exist in this rabbit).
- Rabbit anti-CDK2 polyclonal antibody (Santa-Cruz technology sc-163) diluted 1/200.
- the mouse anti-p21 monoclonal antibody (DAKO, M7202) diluted 1/150.
- PML bodies In the proliferation stage, there are visualized changes in PML bodies because these PML bodies dissociate and degrade: (speekles), proteins are then available in the nucleus to ensure transcription, proliferation, immune responses and anything that requires gene transcription. It has been demonstrated that PML associates with SUMO and HDAC-I (histone deacetylase 1) and that its complex acts on the expression of E2F1 and PML thus acts on the stop of the proliferation by blocking E2F1. So the PML complex / HDAC-I negatively regulates E2F1 expression. PML associated with Rb (pl30) binds to histone deacetylases and blocks E2F1 by binding to chromatin.
- SUMO and HDAC-I histone deacetylase 1
- PML In acute promyelocytic leukemias, PML is truncated and becomes a fusion protein with the retinoic acid receptor. This fusion protein
- PMLRARalpha is due to chromosomal translocation 15/17. It has been reported in the literature a new treatment of this disease by combination of arsenic and retinoic acid to induce cancer cells in apoptosis. The PML protein regulates proliferation in cancers and lymphomas. The inventor has shown by immunoprecipitation the SUMO-PML association in which LIV21 is located.
- LIV21 is phosphorylated by PKC ⁇ and that TPA is a PKC inhibitor.
- MCF7 lines treated with TPA show inhibition of cancer proliferation and cell differentiation and LIV21 is translocated into the nucleus. If a PKC ⁇ -specific inhibitory peptide was used, it is the activity and not the expression of PKC ⁇ that was inhibited.
- E2F4 After 48 hours when proliferation begins, E2F4 has a comparable location; but at 72 h, it disappears from the core (in favor of E2Fl).
- LIV21 which binds to LIV21, actually addresses LIV21 in PML bodies and LIV21 is involved in PML / SUMO / Rb / HDAC-1 complexes.
- LIV21 is physically associated with PML and SUMO in nuclear bodies, by immunoprecipitation and by co-localization in immunocytochemistry ((Rb, pi, and p07 are pocket proteins that have the same binding site).
- Rb suppress cell growth (Fabbro, Regazzi R Bioch
- E2F1 positively controls the cell cycle by transactivating the promoter of the genes responsible for cell proliferation (DNA polymerase alpha, thymidine kinase, DHFR, etc.), whereas E2F4 is described as one of the members of the E2F family that negatively controls the cell proliferation. cycle.
- E2F1 embryonic mammary tissues
- Antigen identification was made in cell lysates by immunoprecipitation.
- the analysis of the physical interaction of different proteins associated with E2F4 and E2F1 was demonstrated by co-immunoprecipitation of protein complexes.
- the study of the complex by ⁇ MACS PROTEIN to MICROBEADS (MILTENYIBIOTEC).
- MILTENYIBIOTEC MILTENYIBIOTEC
- S aureus lysates By addition of S aureus lysates, the A proteins interact with the Fc portion of the specific antibodies and the immune complexes become insoluble, thus recovering them by centrifugation. After breaking the bonds (heating) between AG AC and protein-rich membranes A, a western was made blot.
- Durocher D IA Taylor 5 Sarbassova D, Haire LF, Westcott SL, Jackson SP, Smerdon SJ, Yaffe MB.
- Molecular basis of FHA domain phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. (2000) Mol CeIl, 6 (5) -1169-82.
- Durocher D Jackson SP. The FHA domain. (2002) FEBS Lett, 513 (1): 58-66.
- HSP27 27 kDa heat shock protein
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US8216582B2 (en) * | 2006-06-23 | 2012-07-10 | Alethia Biotherapeutics Inc. | Polynucleotides and polypeptide sequences involved in cancer |
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FR2950076A1 (fr) * | 2009-09-17 | 2011-03-18 | Laurence Faure | Test pharmaco diagnostic ciblant la neuro oncologie et la maladie d'alzheimer |
FR2950075A1 (fr) * | 2009-09-17 | 2011-03-18 | Laurence Faure | Test pharmaco diagnostic ciblant l'oncologie: cancers epidermoides |
CN101963617B (zh) * | 2010-05-26 | 2013-07-10 | 江西中烟工业有限责任公司 | 测定体液中尼古丁的新型间接抑制免疫分析法 |
EP2751134A1 (fr) * | 2011-03-18 | 2014-07-09 | Laurence Faure | Tests dedies a l'oncologie et a la neuro oncologie |
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KR101511737B1 (ko) * | 2012-12-31 | 2015-04-20 | 대한민국 | Sumo1 및 bace1의 결합 억제제를 유효성분으로 함유하는 퇴행성 뇌질환 예방 및 치료용 약학적 조성물 |
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