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WO2000047765A1 - Materiaux et procedes relatifs a la detection de marqueurs de cellules cancereuses - Google Patents

Materiaux et procedes relatifs a la detection de marqueurs de cellules cancereuses Download PDF

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Publication number
WO2000047765A1
WO2000047765A1 PCT/GB2000/000313 GB0000313W WO0047765A1 WO 2000047765 A1 WO2000047765 A1 WO 2000047765A1 GB 0000313 W GB0000313 W GB 0000313W WO 0047765 A1 WO0047765 A1 WO 0047765A1
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Prior art keywords
mitf
nucleic acid
expression
mrna
specific binding
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Colin Ronald Goding
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Marie Curie Cancer Care
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Marie Curie Cancer Care
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    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G01N33/57557—
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to materials and methods involved in the identification and use of a novel cancer cell marker. Particularly, but not exclusively, the present invention provides nucleic acid sequence and polypeptide sequence for Microphthalmia-associated transcription factor (Mitf) mRNA splice variants and methods for detecting said variants in cells such as melanoma cells.
  • Mitf Microphthalmia-associated transcription factor
  • mice bearing mutations in the mi cr ophthalmia gene lack neural crest-derived melanocytes (Stringrimsson et al, (1994) Nat. Genetics 8, 256-263; and Opdecamp et al (1997) Development 124, 2377-2386), and have small eyes due to aberrant formation of the retinal pigment epitheliun (RPE) (Nakayama et al (1998) . Mec. Dev. 70, 155-166) .
  • RPE retinal pigment epitheliun
  • Mitf is mutated in individuals suffering from aardenburg' s syndrome type II which is characterised by varying degrees of abnormal pigmentation and deafness, since melanocytes in the inner ear play an essential role in hearing (Steel and Barkway (1989), Development 107, 453-463). In addition, under some circumstances Mitf can convert fibroblasts to cells expressing melanogenic markers (Tachibana ••et al (1996)
  • Mitf In addition to being expressed in cells of the melanocyte lineage, Mitf is also found in a number of other cell types, including osteoclasts and mast cells. Thus, in addition to defects in the melanocyte lineage, mice lacking functional Mitf also suffer from osteopetrosis, reduced mast cell and natural killer cell numbers .
  • the Mitf mRNA In the melanocyte lineage where Mitf expression is driven by the MITF-M promoter, the Mitf mRNA is present in two differentially spliced forms which give rise to Mitf proteins which differ by an internal 6 amino acids (ACIFPT) located N-terminal to the DNA binding domain (Stringrimsson et al, (1994) Nat. Genetics 8, 256-263) .
  • ACIFPT internal 6 amino acids
  • Mitf (+) The spliced form containing the additional 6 amino acids is termed Mitf (+) and the spliced form with the 6 amino acids absent is termed mitf (-) .
  • mitf (+) The spliced form containing the additional 6 amino acids
  • mitf (-) The spliced form with the 6 amino acids absent is termed mitf (-) .
  • the present inventors have found that in normal cells, the predominant form of Mitf mRNA is Mitf (+) whereas in certain cancer lines tested, the predominant form present of Mitf mRNA is Mitf (-) .
  • the present invention provides the diagnosis of diseases associated with the differentially spliced forms of Mitf by use of specific binding members such as a) nucleic acid molecules hybridisable with a nucleic acid specific to the (+) or (-) forms of Mitf mRNA or cDNA; b) substances comprising an antibody domain with specificity for epitopes or sequences characteristic of either the (+) or (-) forms of Mitf nucleic acid or polypeptide.
  • specific binding members such as a) nucleic acid molecules hybridisable with a nucleic acid specific to the (+) or (-) forms of Mitf mRNA or cDNA; b) substances comprising an antibody domain with specificity for epitopes or sequences characteristic of either the (+) or (-) forms of Mitf nucleic acid or polypeptide.
  • the nucleic acid binding members may take the form of probes for detecting nucleic acid sequences specific for either (+) or (-) forms.
  • the genomic nucleic acid sequence will not distinguish between the two forms.
  • the expressed mRNA will contain nucleic acid sequence specifically characteristic for both forms respectively.
  • the encoded polypeptides will have unique amino acid sequence. These characteristic properties may be utilised in connection with the present invention.
  • the nucleic acid probes preferably comprise sequence having sufficient homology with the distinctive nucleic acid sequences of the Mitf (+) and Mitf (-) forms such that they will hybridize under relatively stringent conditions. The sequence of the probes may be conveniently derived from the sequence shown in Fig.
  • the probes are preferably at least 10 bp in length, more preferably at least 25bp in length, even more preferably at least 40bp in length and most preferably between 20 and 200bp in length.
  • the sequence of the probe as derived from the sequence as shown in Fig. 1, preferably has at least 60% homology with the sequence comprising the Mitf(+ ) sequence, "even more preferably 70%, most preferably 85% homology and particularly preferably 95% homology.
  • nucleic acid sequence having at least 20bp (and preferably no more than lOObp) comprising the sequence as shown in Figure 1 spanning the region indicated as Mitf (+) or its complement sequence for use detecting the Mitf (+) spliced form of Mitf.
  • nucleic acid sequence having at least 20bp (and preferably no more than lOObp) comprising the sequence as shown in Fig. 1 spanning the region indicated as Mitf(+) but excluding this region, for use in detecting the Mitf(-) spliced form of Mitf. It is likely that both spliced forms, i.e.
  • both transcripts, of Mitf (+) and Mitf (-) will be present in the cells under test.
  • the comparative expression of the transcripts alters between normal cells and cancer cells, e.g. melanocytes as compared to melanoma cells. Therefore, the detection of the different spliced forms of Mitf may be followed by comparison of the relative expressed amounts of each form, e.g the amount of mRNA/cDNA or the relative amount, e.g. ratio of expressed polypeptides .
  • Binding of a probe to a target nucleic acid may be measured using any of a variety of techniques at the disposal of those skilled in the art.
  • probes may be radioactively, fluorescently or enzymatically labelled.
  • Other methods not employing labelling of probes include examination of restriction fragment length polymorphisms, amplification by PCR, Rnase cleavage and allele specific oligonucleotide probing.
  • Probing may employ the standard Southern or Northern blotting technique. For instance mRNA or cONA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the cDNA or RNA fragments on the filter and binding or intensity of binding determined. CDNA for probing may be prepared from RNA preparations from cells.
  • Nucleic acid sequences derived from the sequence shown in Fig. 1 and particularly the sequence spanning the Mitf(+) sequence are useful for (a) identifying the presence or absence of the Mitf (+) and Mitf (-) spliced forms or (b) quantifying the transcribed amounts of the Mitf (+) and Mitf (-) spliced forms in a test sample.
  • the present invention provides a method of obtaining nucleic acid of interest, the method including hybridisation of a probe having a sequence including the Mitf (+) sequence as shown in Fig.l, variant mutant or allele thereof or a complementary sequence, to the target nucleic acid, preferably mRNA or cDNA. Hybridization is generally followed by identification and quantification of successful hybridization.
  • mRNA is extracted from cells under test and cDNA is produced therefrom using Reverse transcriptase PCR (RT-PCR) .
  • RT-PCR Reverse transcriptase PCR
  • the produced cDNA will therefore reflect the mRNA forms of Mitf expressed in the test cell.
  • Known techniques can then be carried out using the cDNA to determine the character and quantity of the expressed Mitf forms, for example, hybridization probes, Rnase protection etc.
  • a further preferred method includes in situ hybridization where labelled antisense oligonucleotides of approximately 14 to 100 nucleotides, preferably 20 to 60 nucleotides, even more preferably 40 to 50 nucleotides, are contacted with the cells under test and hybridization to the mRNA transcripts are detected using the label, e.g. radioactivity where the oligonucleotides are end labelled using, for example ⁇ 35 S dATP. Such oligonucleotides may also be used on Northern blots .
  • nucleic acid molecules for use as primers for amplification procedures such as PCR for detecting the presence or absence of the Mitf (+) and Mitf (-) spliced forms or to determine which spliced form is predominantly present in a sample.
  • An oligonucleotide for use in nucleic acid amplification may have about 30 bp or fewer in length.
  • primers are upwards of 14 nucleotides in length, but not more than 18 to 20.
  • Primers may be derived from the sequence shown in Fig. 1 and may, for example, be derived from the sequence indicated in the Figure.
  • the skilled person can determine the different sizes of the resulting amplified nucleic acid molecules by, e.g. gel electrophoresis or standard sequencing protocols. Further, the amounts of the two spliced forms can be quantified by standard methods such as autoradiography, spectrophotometry densitometry and fluorometry
  • the present invention also provides specific binding members comprising an antibody binding domain with specificity for one or more epitopes characteristic of Mitf (+) and Mitf (-) respectively.
  • the encoded polypeptides will also differ in their amino acid sequence. This means that the encoded polypeptides will differ from each other with regard to their immunogenic properties. In other words, the two forms of encoded polypeptide will possess distinguishing epitopes as a result of different amino acid sequence and/or as a result of different folding or conformational properties. These distinguishing immunogenic properties may be utilised in accordance with the present invention.
  • the specific binding members may comprise antibodies, either monoclonal or polyclonal. Alternatively they may comprise derivatives, synthetic analogues or fragments of such antibodies which retain an antibody binding domain with the specificity described above.
  • specific binding member comprising an antibody binding domain hence covers both monoclonal and polyclonal antibodies as well as fragments, derivatives and functional equivalents thereof.
  • the present invention provides a specific binding member which is either (a) specific for the Mitf (+) polypeptide sequence as shown in Fig. 1 or a derivative, allele, mutant or fragment thereof; (b) specific for the Mitf(-) polypeptide sequence as shown in Fig. 1 or a derivative, allele, mutant or fragment thereof; or (c) specific for either a Mitf (+) or Mitf (-) polynucleotide sequence .
  • each spliced variant form, Mitf (+) or Mitf (-) may be determined by standard techniques known to the skilled person, for example, the binding members may be labelled for detection and quantification, e.g. radioactive labelling, fluorescent labelling, enzyme labelling, or further antibody labelling.
  • a further aspect of the present invention provides an antibody able to bind specifically to a Mitf (+) or Mitf (-) polypeptides whose sequences is given in Fig. 1.
  • Such antibodies may be specific in the sense of being able to distinguish between the polypeptides they are able to bind and other human polypeptides for which they have no or substantially no binding affinity (e.g. a binding affinity of about lOOOx worse) .
  • Specific antibodies bind an epitope on the molecule which is either not present or is not accessible on other molecules.
  • a panel of antibodies are used, e.g. different antibodies which are independently specific for each spliced variant form, different reporting labels may be employed for each antibody so that binding of each can be observed and quantified.
  • the present inventors have determined that differential expression of the two spliced variant forms of Mitf (-) and Mitf (+) occur in tumour cell as opposed to normal cells.
  • the inventors have shown the Mitf (-) form is predominantly expressed in tumour cells.
  • detection and quantification of the differential expression of the two spliced variants provides a useful diagnostic tool for determining the presence, type and duration of tumours, for example, melanoma.
  • Methods for determining the concentration of analytes in biological samples from individuals are well known in the art and can be employed in the context of the present invention to determine whether an individual has an elevated level of Mitf (-) expression as compared to the Mitf (+) expression, and so has or is at risk from cancer.
  • the purpose of such analysis may be used for diagnosis or prognosis to assist a physician in determining the severity or likely course of the cancer and/or to optimise treatment of it.
  • Preferred diagnostic methods rely on the detection and quantification of the two spliced variant forms in biological samples such as tissue cells, for example, naevi cells, primary or secondary melanomas, mast cells or osteoclasts and other cell types expressing Mitf mRNA.
  • Assay methods for use in such diagnosis utilise the binding members as described above.
  • the binding members are nucleic acid probes or antibody binding regions, they may be immobilised on a solid support, e.g. at a defined location, to make it easy to manipulate during the assay.
  • the biological sample under test is generally contacted with the binding member under appropriate conditions so that the Mitf spliced variant forms can bind to the member.
  • the occupancy of the binding sites of the binding members can then be determined using a developing agent or agents.
  • the developing agents are labelled (e.g. with radioactive fluorescent or enzyme labels) so that they can be detected using techniques well known in the art.
  • radioactive labels can be detected using a scintillation counter or other radiation counting device, fluorescent labels using a laser and confocal microscope, and enzyme labels by the action of an enzyme label on a substrate, typically to produce a colour change.
  • the developing agent can be used in a competitive method in which the developing agent competes with the spliced variant forms for occupied binding sites of the binding label, or non-competitive method, in which the labelled developing agent binds the variant spliced forms bound by the binding member or to occupied binding sites. Both methods provide an indication of the number of the binding sites occupied by the first spliced variant form, and hence the concentration of the form in the sample, e.g. by comparison with a previous assay for the second variant form.
  • the present invention provides a method of diagnosing a cancer or risk of a cancer, for example, melanoma, in a patient comprising determining the level of expression of the alternative spliced variant forms mitf (+) and Mitf(-) in a biological sample obtained from a patient.
  • a cancer or risk of a cancer for example, melanoma
  • the diagnostic field e.g. making use of binding agents (such as antibodies or nucleic acid sequences) immobilised in small, discrete location (microspots) and/or as arrays on solid supports or on diagnostic chips.
  • the present invention provides a kit comprising a support or diagnostic chip having immobilised thereon one or more binding members capable of specifically binding either of the spliced variant forms Mitf(+) and Mitf(-), optionally in conjunction with other reagents (such as labelled developing reagents) needed to carry out an assay.
  • the present invention also provides a method for screening for candidate compounds capable of modifying expression of Mitf mRNA transcripts, said method comprising exposing cells capable of expressing Mitf mRNA to candidate compounds; detecting expression of Mitf (+) and Mitf (-) forms of Mitf mRNA; and selecting those compounds which modify the ratio of Mitf (+) expression to Mitf (-) expression.
  • a comparison step may in included in this method whereby the ratio of Mitf (-) to Mitf (+) expression is compared to a control step in which the candidate compound has been omitted.
  • a method for screening for compounds capable of inhibiting the relative increased expression of the Mitf (-) transcript as compared to the Mitf (+) transcript comprising contacting a cell in an environment such that it is capable of expressing Mitf mRNA with a candidate compound and detecting the relative expression of Mitf (+) and Mitf (-) forms of Mitf mRNA; and comparing said relative expression with that in a control cell from the same environment in order to determine the inhibition capabilities of the candidate compound.
  • the cells are melanocytes and the candidate compounds have the potential ability to inhibit the MAPkinase pathway directly or indirectly by, for example, inhibition of receptor tyrosine kinase function or RAS or other steps in the MAPkinase signalling cascade.
  • Such a method is useful in determining compounds capable of preventing the switch to increased expression of the Mitf (-) form as opposed to the Mitf (+) form. Such determined compounds may then be utilised in the treatment of cancer cells associated with the increased expression of the Mitf (-) transcript .
  • the present invention also provide a cell system model for use in testing or screening for candidate compounds capable of influencing the relative expression of the Mitf (-) form of Mitf mRNA as compared to the Mitf (+) form of Mitf mRNA, i.e screening for compounds capable of altering the ratio of the Mitf (+) and Mitf (- ) forms as compared to a control system absent of said candidate compound. Such a system may further be used to monitor the progress of a cancer and/or the success of any therapeutics. Such candidate compounds may be of use as therapeutics.
  • Figure 1 DNA and polypeptide sequences corresponding to human (hsMitfna) or mouse (mmMitfna) Mitf. Sequences indicated in bold type correspond to the 6 amino acids uniquely present in the Mitf (+) protein. Sequences underlined indicate bases uniquely present in the Mitf (+) cDNA. The locations of the primers used for the PCR reactions are indicated.
  • Figure 2 A) RT-PCR using RNA derived from the indicated cell lines (melan-a, mouse melanocyte; B16, relatively differentiated mouse melanoma; MM96, human melanoma) .
  • the primers used for the PCR reaction are indicated on the attached Mitf sequence in Figure 1.
  • FIG. 2 B) The melan-a cell line was infected with a retrovirus expressing a 6xHIS tagged version of the constitutively activated form of the MAPkinase kinase MEK (MEK.EE) and two clonal cell lines (MEK1 and MEK2) were isolated. Constitutively active MEK expression was detected using an antibody directed against the 6xHIS tag. MEK1 express a low level of constitutively active MEK and MEK2 at higher level.
  • Figure 2 C) RT PCR using the same primers as in A) and RNA derived from the indicated cell lines. An Mitf (-) cDNA was used as a control. Little or no Mitf (+) mRNA is detected in the MEK1 and MEK2 cell lines.
  • Figure 2 D) Control: PCR using specific Mitf primers and a mixture of the Mitf(+) and Mitf (-) cDNAs as a control for the size of the PCR products expected.
  • M size markers derived from lOObp ladder (BRL) showing positions of 200 and 300bp markers.
  • Dx3, HMB2, MeWo, VUP, A375P and A375M are all human melanoma cell lines provided by Prof. Ian Hart. St Thomas's Hospital London.
  • RNA derived from the melanocyte cell line melan-a together with reverse transcription coupled with the polymerase chain reaction (RT-PCR) using primers corresponding to sequences flanking the differential splice, it is possible to distinguish PCR products corresponding to cDNAs derived from the Mitf (+) and Mitf (-) mRNA.
  • RT-PCR polymerase chain reaction
  • Mek activates MAPkinases such as ERK2, it would appear that constitutive activation of the MAPkinase signalling pathway leading to activation of MEK and ERKs results in cells expressing the Mitf (-) form in preference to the Mitf (+) form.
  • Mitf (-) form of Mitf mRNA in melanoma cells might be accounted for by constitutive signalling by the MAPkinase pathway, resulting either from the constitutive activation of receptor tyrosine kinases, a characteristic of melanomas, or other events leading to constitutive activation of the MAPkinase signalling cascade eg, activating mutations of Ras (reviewed in Chin et al., 1998; Genes Dev. 12, 3467- 3481) .
  • Mitf mRNA or protein may be detected by (i) RT-PCR,
  • melanocytes and melanomas should also be applicable to other cells types in which Mitf is expressed eg. mast cells or osteoclasts.
  • the infected melan-a cells were placed in selection medium containing puromycin to permit the growth of retrovirally infected cells and clonal cell lines established using standard cell cloning techniques.
  • the two derivative cell lines, MEK1 and MEK2 were shown to express low or high levels of constitutively active MEK by western blotting using an anti-His tag antibody (see Fig 2) .
  • Whole cell RNA from all cell lines was isolated using standard techniques and a reverse transcription reaction performed using random primers and AMV reverse transcriptase .
  • A375P has been described as a human low metastatic cell line and A375M as a high metastatic variant of A375P (see for example Hendrix, M.J. et al (1987): A simple quantitative assay for studying the invasive potential of high and low human metastatic variants. Cancer Lett. 38, 137-147. and references therein) .

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Abstract

L'invention porte sur des matériaux et des procédés servant à l'identification et à l'utilisation d'un nouveau marqueur de cellules cancéreuses, sur les séquences d'acides nucléiques et de polypeptides des variantes Mitf(-) et Mitf(+) du Mitf (facteur de transcription associé à la Microphtalmie) de l'ARNm épicé, et sur des procédés de détection de ces variantes particulières dans des cellules telles que celles des mélanomes. Les inventeurs ont découvert que la prédominance dans les cellules normales d'une variante épicée par rapport à une autre s'inversait dans des cellules telles que celles des mélanomes. On obtient ainsi un marqueur de cellules cancéreuses facilitant l'analyse des cellules et pouvant servir à établir des diagnostics ou pronostics aidant le médecin à déterminer la sévérité ou l'évolution probable d'un cancer et d'en optimiser le traitement.
PCT/GB2000/000313 1999-02-08 2000-02-03 Materiaux et procedes relatifs a la detection de marqueurs de cellules cancereuses Ceased WO2000047765A1 (fr)

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FR2869623A1 (fr) * 2004-04-29 2005-11-04 Centre Nat Rech Scient Marqueur predictif de l'evolution des melanomes et ses applications
WO2004060302A3 (fr) * 2002-12-26 2007-08-16 Cemines Llc Methodes et compositions pour le diagnostic, le pronostic, et le traitement du cancer
US7340349B2 (en) 2001-07-25 2008-03-04 Jonathan Bingham Method and system for identifying splice variants of a gene
US7544654B2 (en) 2000-11-10 2009-06-09 The Board Of Trustees Of The Leland Stanford Junior University ψεRACK peptide composition and method for protection against tissue damage due to ischemia
US7833779B2 (en) * 2001-07-25 2010-11-16 Jivan Biologies Inc. Methods and systems for polynucleotide detection
FR2954352A1 (fr) 2009-12-21 2011-06-24 Roussy Inst Gustave Marqueur de predisposition a un cancer

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7544654B2 (en) 2000-11-10 2009-06-09 The Board Of Trustees Of The Leland Stanford Junior University ψεRACK peptide composition and method for protection against tissue damage due to ischemia
US7985726B2 (en) 2000-11-10 2011-07-26 The Board Of Trustees Of The Leland Standford Junior University ψεRACK peptide composition and method for protection against tissue damage due to ischemia
US8592363B2 (en) 2000-11-10 2013-11-26 The Board Of Trustees Of The Leland Stanford Junior University ψεrack peptide composition and method for protection against tissue damage due to ischemia
WO2003004047A1 (fr) * 2001-07-05 2003-01-16 Mitsubishi Pharma Corporation Inducteurs de mort cellulaire pour des mastocytes
US7340349B2 (en) 2001-07-25 2008-03-04 Jonathan Bingham Method and system for identifying splice variants of a gene
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FR2869623A1 (fr) * 2004-04-29 2005-11-04 Centre Nat Rech Scient Marqueur predictif de l'evolution des melanomes et ses applications
WO2005116249A1 (fr) * 2004-04-29 2005-12-08 Centre National De La Recherche Scientifique Marqueur predictif de l'evolution des melanomes et ses applications.
FR2954352A1 (fr) 2009-12-21 2011-06-24 Roussy Inst Gustave Marqueur de predisposition a un cancer
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