WO2006024796A1 - Nouvelles sequences phosphorylees de la phosphatase cdc25b, anticorps diriges contre ces sequences ainsi que leurs utilisations - Google Patents
Nouvelles sequences phosphorylees de la phosphatase cdc25b, anticorps diriges contre ces sequences ainsi que leurs utilisations Download PDFInfo
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- WO2006024796A1 WO2006024796A1 PCT/FR2005/002126 FR2005002126W WO2006024796A1 WO 2006024796 A1 WO2006024796 A1 WO 2006024796A1 FR 2005002126 W FR2005002126 W FR 2005002126W WO 2006024796 A1 WO2006024796 A1 WO 2006024796A1
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- phosphorylated
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- protein
- sequence seq
- serine residue
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the subject of the present invention is novel phosphorylated CDC25CB phosphatase sequences as well as polyclonal or monoclonal antibodies directed against these sequences.
- the subject of the present invention is also the use of these novel phosphorylated sequences, in particular for the implementation of a method for in vitro screening of inhibitory compounds for cell mitosis, namely compounds which inhibit the entry into mitosis of cells. or progression of mitosis.
- CDC25B is a cell cycle regulatory phosphatase essential for the control of mitotic entry and progression of mitosis. It belongs to a family that has three members encoded by different genes (CDC25A, B and C) in mammals.
- the CDC25B protein is expressed and active at the end of the G2 phase of the cell cycle (Baldin et al., 1997, Gabrielli et al., 1996). Its intracellular localization is regulated by NES and NLS sequences (Davezac et al., 2000) and by its interaction with 14-3-3 proteins (Mils et al., 2000, Forrest et al., 2001).
- CDC25B may act as a "starter" of early mitotic events (Nilsson et al., 2000). It may play a role in the initial activation of a CDC2 / cyclin B population at the centrosome before nuclear translocation (Kumagai et al., 1992, Hoffmann et al, 1993). CDC25B activates CDK / cyclin complexes to allow the architectural and biochemical reworkings that are required to enable the process of cell division. Its activity is regulated by the variations of its expression, by its association with regulatory partners and by phosphorylation events. Thus, a signaling cascade leads to the modulation of the catalytic activity of CDC25B involved in regulating the entry into mitosis.
- One of the aims of the invention is to provide a new phosphorylated sequence of CDC25B phosphatase, as well as a new antibody directed against the phosphorylated phosphorylated CDC25B phosphatase epitope.
- One of the aims of the present invention consists in providing a method of in vitro screening of inhibitory compounds for mitosis of cells, in particular the entry into mitosis, said inhibitory compounds being able to be especially used in the context of an anticancer therapy.
- the present invention relates to a peptide sequence characterized in that it comprises or consists of a fragment of at least about 10 amino acids derived from the following sequence SEQ ID NO: 1:
- phosphorylated residue refers to an amino acid carrying a phosphate group.
- the present invention relates to a peptide sequence as defined above, characterized in that it comprises or consists of the following sequence SEQ ID NO: 2:
- sequence SEQ ID NO: 2 corresponds to a fragment of the sequence SEQ ID NO: 1 mentioned above. More exactly, it corresponds to the fragment of SEQ ID NO: 1 delimited from the amino acid in position 4 to the amino acid in position 16.
- the present invention also relates to a peptide sequence as defined above, characterized in that it comprises or consists of one of the following sequences:
- sequence SEQ ID NO: 3 representing the CDC25B1 splice variant of the protein of human origin of the CDC25B phosphatase, whose serine residue in position
- sequence SEQ ID NO: 4 representing a CDC25B2 splice variant of the protein of human origin of CDC25B phospliatase, whose serine residue at position 208 is phosphorylated
- sequence SEQ ID NO: 5 representing a CDC25B3 splice variant of the protein of human origin of the CDC25B phosphatase, whose serine residue in position
- sequence SEQ ID NO: 6 representing a CDC25B4 splice variant of the protein of human origin of the CDC25B phosphatase, whose serine residue at position 259 is phosphorylated
- the present invention also relates to a polyclonal or monoclonal antibody capable of recognizing a peptide sequence as defined above, provided that said antibody does not recognize the sequence SEQ ID NO: 8 in which the serine residue in position 249 does not is not phosphorylated.
- sequence SEQ ID NO: 8 corresponds to the protein sequence of unphosphorylated CDC25B.
- An advantageous polyclonal antibody of the invention is characterized in that it is capable of recognizing the sequence SEQ ID NO: 2 as defined above, provided that said antibody does not recognize the sequence SEQ ID NO: 8 in which the serine residue at position 249 is not phosphorylated.
- Such an antibody directed against the phosphorylated epitope of sequence SEQ ID NO: 2 is generated by immunizing rabbits with said epitope. More specifically, said epitope is covalently coupled with a carrier protein such as hemocyanin, BSA or ovalbumin.
- a carrier protein such as hemocyanin, BSA or ovalbumin.
- the rabbits are then immunized for 3 months (4 injections in total) and the final bleeding allows the recovery of about 50 ml of serum.
- the serum is then doubly purified by affinity on a phosphorylated peptide column and then on a non-phosphorylated peptide column.
- the present invention also relates to a method for preparing a monoclonal antibody as defined above, in particular directed against the peptide sequence SEQ ID NO: 2 as defined above, characterized in that it results from the selection of a hybridoma secreting an antibody directed against the peptide sequence ' as defined above or the selection in an expression library of a complementary DNA encoding all or part of an antibody.
- the present invention also relates to a method for preparing a monoclonal antibody as defined above, in particular directed against the peptide sequence SEQ ID NO: 2 as defined above, characterized in that it comprises the following steps:
- the animal used for the immunization step is especially a mouse.
- Mybites used for fusion come from a mouse.
- the splenocytes used for the fusion come from an animal of the same species as that from which the myelomas originate, namely in particular from a mouse.
- Hybridomas which secrete the antibodies against the peptide sequence SEQ ID NO: 2 are chosen on the basis of the production of antibodies capable of recognizing in an ELISA test the phosphorylated peptide used for the immunization but not the non-phosphorylated peptide.
- the present invention also relates to a method for preparing a monoclonal antibody as defined above, in particular directed against the peptide sequence SEQ ID NO: 2 as defined above, characterized in that it comprises a selection step in an expression library of a cDNA encoding all or part of an antibody.
- the antibodies are selected against the peptide sequence SEQ ID NO: 2 on the basis of their ability to recognize in an ELISA test, by protein transfer (western blot) or by any other appropriate method, the phosphorylated peptide used for the immunization, but not the unphosphorylated peptide.
- the present invention also relates to a pharmaceutical composition characterized in that it contains as active substance a peptide sequence as defined above or an antibody as defined above, in association with a pharmaceutically acceptable carrier.
- the present invention also relates to the use of the peptide sequence as defined above, or of an antibody as defined above, for the preparation of a medicament for the treatment of hyperproliferative diseases such as cancers.
- the present invention also relates to the use of an antibody as defined above, for the implementation of a method for the in vitro detection of mitotic cells, expressing a protein sequence as defined above, among cells in culture or sections of healthy or tumorous tissue.
- the present invention also relates to the use of an antibody as defined above, for the implementation of a method for the in vitro detection of the overexpression of a protein sequence as defined above, in cells in culture or sections of healthy or tumorous tissue, especially in sections of tumors of the breast, lungs or pancreas.
- the present invention also relates to a method for in vitro screening of compounds that inhibit mitosis, namely the entry into mitosis of cells or the progression of mitosis, said inhibitory compounds being able to be used especially in the context of a therapy.
- anticancer agent characterized in that it comprises:
- the selection of the inhibitory compounds by detecting the absence of binding of the antibody as defined above, said antibody being capable of recognizing a peptide sequence as defined above, by an appropriate method, especially using ELISA, protein transfer (Western Blot) or indirect immunofluorescence techniques.
- FIGS. 1A, 1B, 1C, 1D and 1E show the detection of the phosphorylated form of CDC25B on serine 249 by immunofluorescence at different cell stages.
- Figure IA corresponds to the interphase
- Figure IB corresponds to prophase
- Figure IC corresponds to the metaphase
- Figure ID corresponds to anaphase / telophase
- Figure IE corresponds to the Gl phase.
- Figure 2 shows the detection of the phosphorylated form of CDC25B on serine 249 by protein transfer (western blot).
- Mass spectrometry analysis performed on CDC25B detected phosphorylation of the serine residue at position 249.
- the CDC25B protein was purified and then digested with trypsin. MS / MS mass spectrometry analysis revealed the presence of a monophosphorylated peptide. The fragmentation of this peptide allowed the identification of the phosphate group on serine 249.
- Anti-S249P antibodies recognize CDC25B protein in mitotic cells.
- HeLa cells were fixed and used to perform immunofluorescence analysis with these antibodies. The cells were also stained with 4'-6-diamino-2-phenylindole (DAPI) to locate the nucleus.
- DAPI 4'-6-diamino-2-phenylindole
- the images presented in Figure 1 are representative of observations on a large number of cells. They indicate that the CDC25B protein phosphorylated on serine 249 (SEQ ID NO: 2) is accumulated very abundantly in mitotic cells, in particular at the spindle poles. This labeling is abolished when a competition is carried out with the test with the phosphorylated peptide having served for the immunization but not with the non-phosphorylated peptide (MEVEELSPLALGR) or with an unrelated phosphorylated peptide.
- the CDC25B protein was purified from cultured cells and then analyzed by western blot with the antibodies against CDC25B phosphorylated phosphatase at serine 249. As shown in Figure 2, the CDC25B protein is phosphorylated in vivo at this site. Treatment with lambda phosphatase abolishes this phosphorylation. The detection is eliminated by competition with the phosphorylated peptide used for immunization.
- a - Mitotic cell marker
- the detection of the phosphorylation of the phosphorylated form of CDC25B makes it possible to detect the presence of mitotic cells on cells in culture or on tumor sections.
- the phosphorylated form of CDC25B on serine 249 is present in the mitotic cell. Its location is nuclear in prophase, then it is concentrated in metaphase on the two mitotic hemi-spindles before invading the equatorial plane in telophase, then disappear at the end of mitosis.
- the polyclonal or monoclonal antibodies directed against this phosphorylated form of CDC25B therefore allow the detection of any mitotic cell expressing CDC25B phosphatase.
- This detection of mitotic cells can be carried out on cells in fixed culture or on sections of healthy or tumoral tissues, using indirect immunofluorescence or immunocytochemistry techniques.
- the detection of mitotic cells by this method is thus a new tool at the disposal of cytologists and anatomo-pathologists.
- CDC25B The expression of CDC25B is variable depending on the tissues. It has been shown that tumors (breast, lung, pancreas, ...) overexpress this protein. In the case of pancreatic tumors, tumor growth is dependent on the expression and function of CDC25B (Guo et al, 2004).
- New pharmacological agents capable of targeting CDC25B are currently being developed by the industry (Prévost et al., 2003). It is therefore essential to take into account the level of expression of CDC25B phosphatase in order to determine the relevance of the use of such a treatment.
- the use of antibodies directed against the phosphorylated form of CDC25B on serine 249 reveals the mitotic (and probably active) form of this phosphatase. Its detection (by immunocytochemistry) in biopsies or surgical specimens after surgical excision provides information likely to guide the therapeutic choice by possibly posing, if CDC25B is overexpressed, the indication of a use of inhibitors of CDC25 phosphatases.
- the search for molecules interfering with the cell cycle may use the antibodies according to the invention to evaluate the inhibition of entry into mitosis.
- An active search for molecules inhibiting progression in the cell cycle is currently being conducted by many pharmaceutical groups.
- Demonstration of a mitotic cell marker is a tool of choice for simply exploring, in high-throughput screens, the ability of molecules to inhibit entry and progression into mitosis.
- Antibodies against the phosphorylated form of CDC25B can meet this need. They can thus be used in immunocytochemistry, flow cytometry or any other suitable method for detecting the phosphorylated CDC25B protein.
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2007528921A JP2008515779A (ja) | 2004-08-25 | 2005-08-23 | 新規リン酸化cdc25bホスファターゼ配列、その配列に対する抗体並びにその利用 |
EP05798193A EP1781696A1 (fr) | 2004-08-25 | 2005-08-23 | Nouvelles sequences phosphorylees de la phosphatase cdc25b, anticorps diriges contre ces sequences ainsi que leurs utilisations |
CA002577950A CA2577950A1 (fr) | 2004-08-25 | 2005-08-23 | Nouvelles sequences phosphorylees de la phosphatase cdc25b, anticorps diriges contre ces sequences ainsi que leurs utilisations |
US11/660,703 US20080219987A1 (en) | 2004-08-25 | 2005-08-23 | Novel Phosphorylated Phosphatase Cdc25b Sequences, Antibodies Directed Against Said Sequences and Uses Thereof |
Applications Claiming Priority (2)
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FR0409080A FR2874619B1 (fr) | 2004-08-25 | 2004-08-25 | Nouvelles sequences phosphorylees de la phosphatase cdc25b, anticorps diriges contre ces sequences ainsi que leurs utilisations |
FR0409080 | 2004-08-25 |
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WO2006024796A1 true WO2006024796A1 (fr) | 2006-03-09 |
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PCT/FR2005/002126 WO2006024796A1 (fr) | 2004-08-25 | 2005-08-23 | Nouvelles sequences phosphorylees de la phosphatase cdc25b, anticorps diriges contre ces sequences ainsi que leurs utilisations |
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US (1) | US20080219987A1 (fr) |
EP (1) | EP1781696A1 (fr) |
JP (1) | JP2008515779A (fr) |
CA (1) | CA2577950A1 (fr) |
FR (1) | FR2874619B1 (fr) |
WO (1) | WO2006024796A1 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2923297A1 (fr) * | 2007-11-02 | 2009-05-08 | Centre Nat Rech Scient | Methode de detection de cellules mitotiques |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2002038143A2 (fr) * | 2000-11-07 | 2002-05-16 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Amelioration de l'efficacite et de la securite d'une therapie genotoxique par modulation de la proteine kinase mapk p38 |
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2004
- 2004-08-25 FR FR0409080A patent/FR2874619B1/fr not_active Expired - Fee Related
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2005
- 2005-08-23 US US11/660,703 patent/US20080219987A1/en not_active Abandoned
- 2005-08-23 EP EP05798193A patent/EP1781696A1/fr not_active Withdrawn
- 2005-08-23 JP JP2007528921A patent/JP2008515779A/ja active Pending
- 2005-08-23 CA CA002577950A patent/CA2577950A1/fr not_active Abandoned
- 2005-08-23 WO PCT/FR2005/002126 patent/WO2006024796A1/fr active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2002038143A2 (fr) * | 2000-11-07 | 2002-05-16 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Amelioration de l'efficacite et de la securite d'une therapie genotoxique par modulation de la proteine kinase mapk p38 |
Non-Patent Citations (5)
Title |
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BALDIN V ET AL: "Phosphorylation of human CDC25B phosphatase by CDK1-cyclin A triggers its proteasome-dependent degradation.", THE JOURNAL OF BIOLOGICAL CHEMISTRY. 26 DEC 1997, vol. 272, no. 52, 26 December 1997 (1997-12-26), pages 32731 - 32734, XP002309641, ISSN: 0021-9258 * |
BALDIN V ET AL: "PKB/Akt phosphorylates the CDC25B phosphatase and regulates its intracellular localisation", BIOLOGY OF THE CELL, ELSEVIER, PARIS, FR, vol. 95, no. 8, November 2003 (2003-11-01), pages 547 - 554, XP002277362, ISSN: 0248-4900 * |
DUTERTRE S ET AL: "PHOSPHORYLATION OF CDC25B BY AURORA-A AT THE CENTROSOME CONTRIBUTES TO THE G2-M TRANSITION", JOURNAL OF CELL SCIENCE, CAMBRIDGE UNIVERSITY PRESS, LONDON, GB, vol. 117, no. 12, 15 May 2004 (2004-05-15), pages 2523 - 2531, XP009036107, ISSN: 0021-9533 * |
GABRIELLI BRIAN G ET AL: "Hyperphosphorylation of the N-terminal domain of Cdc25 regulates activity toward cyclin B1/Cdc2 but not cyclin A/Cdk2", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 272, no. 45, 7 November 1997 (1997-11-07), pages 28607 - 28614, XP002173983, ISSN: 0021-9258 * |
LAMMER C ET AL: "The cdc25B phosphatase is essential for the G2/M phase transition in human cells.", JOURNAL OF CELL SCIENCE. AUG 1998, vol. 111 ( Pt 16), August 1998 (1998-08-01), pages 2445 - 2453, XP002309642, ISSN: 0021-9533 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2923297A1 (fr) * | 2007-11-02 | 2009-05-08 | Centre Nat Rech Scient | Methode de detection de cellules mitotiques |
WO2009103862A2 (fr) * | 2007-11-02 | 2009-08-27 | Centre National De La Recherche Scientifique | Methode de detection de cellules mitotiques |
WO2009103862A3 (fr) * | 2007-11-02 | 2009-10-15 | Centre National De La Recherche Scientifique | Methode de detection de cellules mitotiques |
Also Published As
Publication number | Publication date |
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US20080219987A1 (en) | 2008-09-11 |
FR2874619A1 (fr) | 2006-03-03 |
JP2008515779A (ja) | 2008-05-15 |
CA2577950A1 (fr) | 2006-03-09 |
FR2874619B1 (fr) | 2006-11-17 |
EP1781696A1 (fr) | 2007-05-09 |
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