WO2008015942A1 - Agent thérapeutique contre le neuroblastome, procédé de criblage à la recherche de l'agent thérapeutique et procédé de détermination du pronostic d'un neuroblastome - Google Patents

Agent thérapeutique contre le neuroblastome, procédé de criblage à la recherche de l'agent thérapeutique et procédé de détermination du pronostic d'un neuroblastome Download PDF

Info

Publication number
WO2008015942A1
WO2008015942A1 PCT/JP2007/064597 JP2007064597W WO2008015942A1 WO 2008015942 A1 WO2008015942 A1 WO 2008015942A1 JP 2007064597 W JP2007064597 W JP 2007064597W WO 2008015942 A1 WO2008015942 A1 WO 2008015942A1
Authority
WO
WIPO (PCT)
Prior art keywords
neuroblastoma
shf
gene
expression level
therapeutic agent
Prior art date
Application number
PCT/JP2007/064597
Other languages
English (en)
Japanese (ja)
Inventor
Akira Nakagawara
Original Assignee
Hisamitsu Pharmaceutical Co., Inc.
Chiba-Prefecture
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hisamitsu Pharmaceutical Co., Inc., Chiba-Prefecture filed Critical Hisamitsu Pharmaceutical Co., Inc.
Publication of WO2008015942A1 publication Critical patent/WO2008015942A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic 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
    • G01N33/57407—Specifically defined cancers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a therapeutic agent for neuroblastoma, a screening method thereof, and a method for determining prognosis of neuroblastoma.
  • Neuroblastoma is a tumor that arises from the sympathetic nervous system (paraspinal sympathetic nerve trunk and adrenal medullary nerve cells), and is most common in malignant solid tumors in children.
  • the most common sites of neuroblastoma are the adrenal gland, the retroperitoneum, the posterior mediastinum, the neck, the sympathetic ganglion of the pelvis, and the midline of the abdominal cavity.
  • Neuroblastoma is extremely interesting clinically! / In cases where the onset age is less than 1 year of age, neuroblastoma has obvious spontaneous regression and maturation, while onset age is more than 1 year of age. In these cases, metastasis and growth of the tumor progress rapidly, resulting in a poor prognosis that is difficult to treat.
  • TrkA is present in the cell membrane, stimulated by nerve growth factor NGF, and transmits the stimulation as a signal via TrkA and various proteins in the cell. As a result, TrkA is thought to cause suppression of cell differentiation and proliferation. However, the molecular mechanism involved in TrkA in neuroblastoma remains unclear.
  • the present inventors have developed novel genes from a plurality of neuroblastoma cDNA libraries in order to develop therapeutic methods for neuroblastoma, to identify genes involved in neuroblastoma development and biological characteristics thereof.
  • Patent Documents 1 to 5 and Non-Patent Document 2 have been isolated.
  • Patent Document 1 Pamphlet of International Publication No. 01/66719
  • Patent Document 2 Pamphlet of International Publication No. 01/66733
  • Patent Document 3 International Publication No. 02/97093 Pamphlet
  • Patent Document 4 International Publication No. 02/103017 Pamphlet
  • Patent Document 5 Pamphlet of International Publication No. 2004/39975
  • Non-patent literature l Nakagawara, Med. Pediatr. Oncol. 31, 1 13 (1998)
  • Non-patent literature 2 OhiraM et al., Oncogene, 22, 5525-5536 (2003) Disclosure of the invention
  • an object of the present invention is to provide a therapeutic agent for neuroblastoma and a screening method thereof.
  • TrkA in neuroblastoma
  • expression of TrkA in neuroblastoma is currently considered as one candidate for determining the prognosis of the disease.
  • TrkA it is unclear how TrkA is involved in determining good and poor prognosis of neuroblastoma.
  • the prognosis may not always be good if there is an abnormality in the TrkA intracellular signaling pathway. Therefore, an object of the present invention is to clarify the molecular mechanism that controls the prognosis of neuroblastoma and to provide a method for determining the prognosis of neuroblastoma.
  • the inventors of the present invention mediate the interaction between signal molecules, and a gene encoding Shf called adapter protein is highly expressed in a good prognosis group. Found that. Furthermore, the present inventors have found that the expression level of Shf gene is highly correlated with the prognosis of neuroblastoma, and in addition, is highly correlated with the expression level of TrkA gene. In addition, Shf and TrkA The inventors have shown that they co-localize and interact with each other. This suggests that Shf binds to TrkA to regulate TrkA signaling and may be involved in the determination of neuroblastoma prognosis. Based on the above findings, the present inventors have completed the present invention.
  • the present invention includes a step of culturing a cell under each condition in the presence and absence of a test compound, and a step of measuring the expression level of the Shf gene in each cultured cell. If the expression level of the Shf gene in the cells cultured in the presence of the test compound is higher than the expression level of the Shf gene in the cells cultured in the absence of the test compound, the test compound is And a method for screening a therapeutic agent for neuroblastoma, comprising the step of determining that the therapeutic agent is a neuroblastoma.
  • the screening method of the present invention is based on the knowledge newly discovered by the present inventor that the expression level of Shf gene strongly correlates with good prognosis and poor prognosis of neuroblastoma. This makes it possible to develop therapeutic agents for improving the prognosis of neuroblastoma.
  • the screening method of the present invention also includes a step of culturing cells in the presence and absence of a test compound, and the Shf gene and TrkA gene in each cultured cell.
  • the expression level of Shf gene and TrkA gene in cells cultured in the presence of the test compound !, Shf in cells cultured in the absence of the test compound And a step of determining the test compound as a therapeutic agent for neuroblastoma when the expression level of the gene and the TrkA gene is higher.
  • the screening method of the present invention also comprises a step of culturing cells in the presence and absence of a test compound, and the mutual relationship between Shf and TrkA in each cultured cell.
  • the interaction force of Shf and TrkA in the cells cultured in the presence of the test compound S, Shf and in the cells cultured in the absence of the test compound And a step of determining the test compound as a therapeutic agent for neuroblastoma when the interaction is stronger than TrkA interaction.
  • the screening method of the present invention It applies the molecular mechanism that Shf and TrkA interact with each other. This molecular mechanism has been newly discovered by the present inventor, which enables the control of Trk A signaling by Shf and the new molecular mechanism based on the new molecular mechanism. Development is possible.
  • the cell to be used in the screening method of the present invention is a cell derived from a clinical sample of human neuroblastoma.
  • the cell is derived from a clinical sample of human neuroblastoma having a poor prognosis. It is preferable. This makes it possible to develop therapeutic agents for improving the prognosis of human neuroblastoma, particularly for the treatment of patients with poor prognosis.
  • the present invention provides a therapeutic agent for neuroblastoma comprising a vector having a nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 1.
  • the present invention also relates to the use of a vector having a nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 1 for the manufacture of a therapeutic agent for neuroblastoma, and to a neuroblastoma patient described in SEQ ID NO: 1.
  • a method for treating neuroblastoma comprising a step of administering a vector having a nucleic acid comprising a base sequence.
  • the present invention provides a therapeutic agent for neuroblastoma comprising a vector having a nucleic acid encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2.
  • the present invention also relates to the use of a vector having a nucleic acid encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 for the manufacture of a therapeutic agent for neuroblastoma, and to a neuroblastoma patient, SEQ ID NO: 2
  • a method for treating neuroblastoma comprising the step of administering a vector having a nucleic acid encoding a protein comprising the amino acid sequence described in 1.
  • the base sequence described in SEQ ID NO: 1 corresponds to the Shf gene
  • the amino acid sequence described in SEQ ID NO: 2 corresponds to Shf.
  • the Shf gene and Shf GenBank Accession No. is NM138356.
  • the prognosis improvement of neuroblastoma disease can be expected by the expression of Shf gene and the enhancement of Shf function.
  • the therapeutic agent of the present invention further includes a vector having a nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 3 or a vector having a nucleic acid encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4.
  • the base sequence described in SEQ ID NO: 3 corresponds to the Trk A gene
  • the amino acid sequence described in SEQ ID NO: 4 corresponds to TrkA (GenBank Accession No .: NP002529).
  • TrkA GenBank Accession No .: NP002529
  • the present invention further comprises a step of measuring the expression level of Shf gene in a clinical sample of human neuroblastoma with unknown prognosis, and the expression level of Shf gene with a good prognosis and a poor prognosis.
  • a method for determining the prognosis of human neuroblastoma comprising the step of comparing the expression level of the Shf gene in a clinical sample of neuroblastoma.
  • the determination method of the present invention is based on the finding that the expression level of the Shf gene is strongly correlated with the survival rate of patients with neuroblastoma, that is, with a good prognosis and poor prognosis of neuroblastoma.
  • Such a molecular mechanism has been newly discovered by the present inventor, and this makes it possible to determine the prognosis of human neuroblastoma whose prognosis is unknown.
  • the determination method of the present invention comprises a step of measuring the expression level of Trk A gene in a clinical sample of human neuroblastoma of unknown prognosis, and the expression level of TrkA gene is determined with good prognosis and poor prognosis. It is preferable to further comprise a step of comparing the expression level of the TrkA gene in a clinical sample of human neuroblastoma.
  • the determination method of the present invention is an application of the knowledge of the present inventors that the expression level of Shf gene is highly correlated with the prognosis of neuroblastoma, and also highly correlated with the expression level of TrkA gene. It is. By analyzing the expression level of the TrkA gene in addition to the analysis of the Shf gene, the prognosis of human neuroblastoma with an unknown prognosis can be determined with higher reliability.
  • the screening method of the present invention it is possible to develop a therapeutic agent for improving the prognosis of neuroblastoma, which has a different mechanism of action from the past.
  • the therapeutic agent of the present invention can be expected to improve the prognosis of neuroblastoma disease.
  • the determination method of the present invention makes it possible to determine the prognosis of a human neuroblastoma whose prognosis is unknown. By combining the determination method of the present invention with the screening method and / or therapeutic agent, it becomes possible to develop a therapeutic agent and select a therapeutic method in accordance with the prognosis good and poor prognosis of neuroblastoma.
  • FIG. 1 Compared to electrophoretic photographs showing the measurement results of the expression level of Shf mRNA in clinical samples of neuroblastoma (eight samples each with good prognosis and poor prognosis) by semi-quantitative RT-PCR.
  • FIG. 1 Compared to electrophoretic photographs showing the measurement results of the expression level of Shf mRNA in clinical samples of neuroblastoma (eight samples each with good prognosis and poor prognosis) by semi-quantitative RT-PCR.
  • FIG. 2 is a graph showing the correlation between Shf mRNA expression level and prognosis.
  • FIG. 3 is a graph showing the correlation between expression levels of TrkA mRNA and Shf mRNA.
  • FIG. 4 is a diagram corresponding to an electrophoretic photograph showing the measurement result of the expression level of Shf mRNA in a normal tissue by a semiquantitative RT-PCR method.
  • FIG. 5 is a diagram corresponding to a stained image showing expression of Shf mRNA by an in situ hybridization method.
  • sp spinal cord
  • DRG dorsal root ganglion
  • dien C e phalon diencephalon.
  • FIG. 6 is a diagram corresponding to a stained image showing the localization of Shf and TrkA in rat pheochromocytoma PC 12 cells by immunostaining experiments.
  • FIG. 7 is a view corresponding to a Western plot image showing the interaction between Shf and TrkA by a coimmunoprecipitation experiment.
  • Shf and TrkA expression constructs were cotransfected into H1299 cells.
  • Anti-Shf antibody and anti-TrkA antibody were used for immunoprecipitation and Western blot analysis.
  • FIG. 8 is a schematic diagram showing a signal transduction mechanism model of Shf and TrkA.
  • the therapeutic drug screening method in the present embodiment includes a screening method for the first and second therapeutic drugs using the expression level of the Shf gene as an index, and a third treatment using the interaction between Shf and TrkA as an index. There are drug screening methods.
  • the first therapeutic drug screening method involves the step of culturing cells in the presence and absence of a test compound, and the expression level of Shf gene in each cultured cell.
  • the measurement step and the expression level S of Shf gene in cells cultured in the presence of the test compound are higher than the expression level of Shf gene in cells cultured in the absence of the test compound, Determining the test compound as a therapeutic agent for neuroblastoma.
  • the expression level of the gene means the expression level of mRNA that is a transcription product of the gene and / or Indicates the expression level of the protein which is the translation product.
  • the measurement of mRNA expression can be performed using a measurement system known to those skilled in the art. Specifically, quantitative RT-PCR, quantitative real-time RT-PCR, quantitative northern blot And the like, and quantitative ribonuclease protection method.
  • the protein expression level may be measured using a measurement system known to those skilled in the art, for example, quantitative Western blotting and ELISA.
  • the expression level of the target gene such as the Shf gene is standardized using the expression level of mRNA and / or protein such as GADPH, which is a housekeeping gene, and beta-actin.
  • GADPH which is a housekeeping gene
  • beta-actin a housekeeping gene
  • the expression level of the target gene and / or control gene in a plurality of samples collected from the same target force and / or the same sample may be measured, and the expression level may be obtained from the average value of each.
  • a test compound in which the expression level of the Shf gene in the presence of the test compound is larger than the expression level of the Shf gene in the absence of the test compound can be determined as a therapeutic agent for neuroblastoma.
  • the therapeutic agent is most preferably an agent that improves the prognosis of neuroblastoma, but any agent that improves the prognosis is suitable for the purpose of the present invention.
  • the expression level of the TrkA gene which is a candidate gene involved in the prognosis determination of neuroblastoma disease, in addition to the expression level of the Shf gene.
  • expression levels of genes that are differently expressed between two subsets of a well-known group and a poor-prognosis group, which are conventionally known may be analyzed simultaneously.
  • the cells used for screening may be cultured cells such as H1299 derived from human lung cancer cells, PC 12 derived from rat adrenal pheochromocytoma, or may be cultured cells derived from neural tissue. Further, it is more preferable that the cultured cell derived from neuroblastoma is a cell derived from a preferred clinical sample of human neuroblastoma, more preferably a cell derived from a clinical neuron of human neuroblastoma having a poor prognosis. .
  • Test compounds include, but are not limited to, low molecular weight compounds, peptides, proteins, nucleic acids (DNA, RNA, PNA) and the like. Also, for screening, any screen Compound library may be used. It should be noted that cell culture conditions and test compound administration conditions can be appropriately adjusted by those skilled in the art.
  • a third method for screening for therapeutic agents is a step of culturing cells in each condition in the presence and absence of a test compound, and a step of measuring the interaction of Shf and TrkA in each cultured cell. And the interaction force S of Shf and TrkA in the cells cultivated in the presence of the test compound, and the interaction force Shf and TrkA in the cells cultivated in the absence of the test compound, And determining the test compound as a therapeutic agent for neuroblastoma.
  • Shf and TrkA expressed in cells are preferably endogenous, but in this case, Shf and TrkA can be expressed by tags such as GST and HA. May be labeled with a fluorescent protein which may be fused. Interaction refers to direct and / or indirect interaction between proteins, where Shf and Tr kA form a complex or are in close proximity to be functionally linked. It means doing.
  • a system for measuring protein interactions known to those skilled in the art can be used. Specifically, the measurement by co-immunoprecipitation method or fluorescence resonance energy transfer (FRET) is applied. Measurement and the like. The obtained quantitative measurement value is compared between the presence and absence of the test compound to determine the utility of the test compound as a therapeutic agent for neuroblastoma.
  • FRET fluorescence resonance energy transfer
  • the therapeutic agent for neuroblastoma which is an embodiment of the present invention, includes a vector having the Shf gene (SEQ ID NO: 1).
  • the therapeutic agent comprises a vector having a nucleic acid encoding Shf (SEQ ID NO: 2).
  • the therapeutic agent preferably further comprises a vector having a TrkA gene (SEQ ID NO: 3) or a vector having a nucleic acid encoding TrkA (SEQ ID NO: 4).
  • Vectors can be prepared based on DNA or RNA viruses. Any virus vector such as a MoMLV vector, a herpes virus vector, an adenovirus vector, an AAV vector, an HIV vector, an SIV vector, or a Sendai virus vector may be used. In addition, one or more of the viral protein component proteins are replaced with a heterologous virus component protein, or a part of the base sequence constituting the genetic information is replaced with a heterologous virus base sequence.
  • a pseudo-type virus vector can also be used in the present invention.
  • viruses having a host range other than humans can be used as virus vectors as long as they have a therapeutic effect.
  • a vector other than a virus a complex of calcium phosphate and nucleic acid, a liposome, a cationic lipid complex, a Sendai virus ribosome, a polymer carrier having a polycation as a main chain, and the like can be used.
  • the expression cassette used for the expression of the gene in the vector can be used without particular limitation as long as it can express the gene in the target cell.
  • One skilled in the art can readily select such an expression cassette.
  • it is an expression cassette capable of gene expression in animal-derived cells, more preferably an expression cassette capable of gene expression in mammal-derived cells, and particularly preferably in human-derived cells. This is an expression cassette capable of gene expression.
  • the gene promoter used in the expression cassette is, for example, adenovirus, cytomegalovirus, human immunodeficiency virus, simian virus 40, rous sarcoma virus, herpes simplex virus, mouse leukemia virus, symbis virus, hepatitis A Virus, Hepatitis B virus, Hepatitis C virus, Papilloma virus, Human T-cell leukemia virus, Influenza virus, Japanese encephalitis virus, JC virus, Parvovirus B19, Poliovirus-derived promoter, albumin, SR a, promoters derived from mammals such as heat shock proteins and elongation factors, chimeric promoters such as CAG promoters, promoters whose expression is induced by tetracycline, steroids, and the like.
  • adenovirus cytomegalovirus
  • human immunodeficiency virus simian virus 40
  • rous sarcoma virus herpes simplex virus
  • the method of determining the prognosis of neuroblastoma in the present embodiment includes a step of measuring the expression level of Shf gene in a clinical sample of human neuroblastoma with an unknown prognosis, and the expression level of Shf gene with a good prognosis, and Shf remains in clinical samples of human neuroblastoma with poor prognosis And a step of comparing with the expression level of the gene.
  • the prognosis is determined based on the knowledge of the present invention that the prognosis of neuroblastoma is good when the expression level of the Shf gene is high.
  • the expression level of Shf gene in clinical samples of human neuroblastoma with good prognosis is a statistically processed measurement of the expression level obtained from multiple cases with good prognosis (good prognosis group). However, the same is true for the expression level of the Shf gene in clinical samples of human neuroblastoma with poor prognosis.
  • the expression level of Sh f gene in clinical samples of human neuroblastoma with unknown prognosis is the same as the expression of Shf gene in clinical samples of human neuroblastoma with good prognosis. If the amount is higher than the dose and / or statistically falls within the distribution range of the good prognosis group in comparison with the expression level obtained from the good prognosis group, the prognosis of the subject is determined to be good. can do. In addition, judgment of poor prognosis can be performed in the same way as judgment of good prognosis.
  • good prognosis refers to a state of a neuroblastoma where the tumor is localized or has become a regression or benign sympathetic ganglion cell, N — Judging from myc and other tumor markers, the grade is low.
  • the N-myc gene is usually only one per haploid in normal cells and neuroblastomas with good prognosis, whereas in neuroblastoma with poor prognosis, it is amplified several tens of times. Is an oncogene.
  • stage 1, 2 or 4 s based on the international neuroblastoma staging age of onset is less than age, and survives 5 years or more after surgery without recurrence.
  • this is not limited to such a specific example, in which N-myc amplification is not recognized as a good prognosis in human neuroblastoma.
  • the term "poor prognosis” as used herein refers to a state in which tumor progression is observed among neuroblastomas, and is highly malignant as judged from N-myc and other tumor markers. It is judged.
  • humans are those whose stage 3 or 4 has an onset age of 1 year or more, died within 3 years after surgery, and N-myc amplification was observed in clinical tissues.
  • Force S which is a poor prognosis in neuroblastoma, is not limited to such a specific example.
  • the method for determining the prognosis of neuroblastoma includes the expression level of the Shf gene, Ability to analyze the expression level of the TrkA gene, which is a candidate gene for determining the prognosis of neuroblastoma disease.
  • the expression levels of genes that are differently expressed between two subsets of a well-known group and a poor-prognosis group, which are conventionally known may be analyzed simultaneously.
  • Superscript II reverse transcriptase was purchased from LifeTechnologies. Random primers were purchased from Takara Sake Brewery. TaqMan (registered trademark) Universal PCR Master Mix was purchased from Perkin—Elmer Applied Biosystems. Shf probed primers were purchased from Applied Biosystems. Anti-Shf antibody was purchased from MBL. Anti-Trk antibody was purchased from SANTA CRUZ BIOTECHNOLOGLY, and anti-Tubul in antibody was purchased from Neo MARKERS.
  • the semi-quantitative RT-PCR method, immunostaining method and immunoprecipitation method are the same as those described in Hanamoto et al., J. Biol. Chem., 280: 16665-166675, 2005.
  • the experimental system was optimized.
  • the experimental system was optimized.
  • Example 1 Expression analysis by RT-PCR method
  • Total RNA was prepared from clinical samples of human neuroblastoma with good prognosis and poor prognosis, and the expression level of Shf mRNA was analyzed by semi-quantitative RT-PCR using these total RNAs as templates. The results are shown in Fig. 1. It was confirmed that the Shf gene was highly expressed in the good prognosis group, suggesting that there is a functional relationship between the expression level of the Shf gene and the prognosis of neuroblastoma.
  • FIG. 4 shows the results of comparing the expression level of Shf mRNA in each normal tissue by semi-quantitative RT-PCR. High expression of Shf mRNA was observed in the brain, cerebellum and fetal brain. Furthermore, Fig. 5 shows the results of analysis by the in situ hybridization method. Embryonic 13. Examination of Shf mRNA expression tissue on day 5 revealed expression in the spinal cord, dorsal root ganglia and diencephalon. Therefore, it was suggested that the Shf gene is expressed in the nervous system and is involved in the maintenance and differentiation of neural tissues.
  • TrkA is transmembrane type Considering that it is a receptor for Sf, it was suggested that Sh f and TrkA co-localize in the cytoplasm and / or cell membrane and interact functionally with each other.
  • H1299 cells were cotransduced with Shf and TrkA expression constructs. After the cultivation, the cell suspension was fractionated into a soluble fraction and an insoluble fraction, and the obtained soluble fraction was subjected to an immunoprecipitation experiment. Both Shf and TrkA were detected in the soluble fraction.
  • Figure 7 shows the analysis results of the coimmunoprecipitation experiment. From this result, it was shown that Shf and TrkA co-expressed together in H1299 cells form an immune complex.
  • FIG. 8 is a schematic diagram showing a signal transduction mechanism model of Shf and TrkA.
  • NGF nerve growth factor
  • TrkA cell membrane-like nerve growth factor receptor
  • the signal is transmitted to the inside of the cell and to the nucleus, and responses such as nerve cell differentiation and cell growth arrest occur.
  • Shf is involved in the NGF / TrkA signal by binding to TrkA and is further involved in determining the prognosis of neuroblastoma by transmitting the signal downstream. The possibility of! / Was suggested.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Neurology (AREA)
  • Toxicology (AREA)
  • Epidemiology (AREA)
  • Neurosurgery (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un procédé de criblage à la recherche d'un agent thérapeutique contre le neuroblastome comprenant les étapes suivantes : cultiver une cellule en présence ou en l'absence d'un composé à tester ; mesurer le taux d'expression d'un gène Shf dans une cellule cultivée ; et déterminer que le composé est un agent thérapeutique contre le neuroblastome quand le taux d'expression du gène Shf dans une cellule cultivée en présence du composé est plus élevé que celui dans une cellule cultivée en l'absence du composé.
PCT/JP2007/064597 2006-07-31 2007-07-25 Agent thérapeutique contre le neuroblastome, procédé de criblage à la recherche de l'agent thérapeutique et procédé de détermination du pronostic d'un neuroblastome WO2008015942A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2006208812A JP2008029296A (ja) 2006-07-31 2006-07-31 神経芽種の治療薬及びそのスクリーニング方法、並びに、神経芽種の予後の判定方法
JP2006-208812 2006-07-31

Publications (1)

Publication Number Publication Date
WO2008015942A1 true WO2008015942A1 (fr) 2008-02-07

Family

ID=38997122

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2007/064597 WO2008015942A1 (fr) 2006-07-31 2007-07-25 Agent thérapeutique contre le neuroblastome, procédé de criblage à la recherche de l'agent thérapeutique et procédé de détermination du pronostic d'un neuroblastome

Country Status (2)

Country Link
JP (1) JP2008029296A (fr)
WO (1) WO2008015942A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001066733A1 (fr) * 2000-03-07 2001-09-13 Chiba-Prefecture Sequences d'acides nucleiques presentant une expression accrue dans le neuroblastome benin par rapport au neuroblastome humain acritique
WO2001066719A1 (fr) * 2000-03-07 2001-09-13 Chiba-Prefecture Nouveau gene clone en neuroblastome humain et nouveaux fragments de gene
WO2002003071A2 (fr) * 2000-07-05 2002-01-10 Pangene Corporation Modulateurs de l'activite de la proteine trk, et compositions et methodes d'utilisation
WO2002097093A1 (fr) * 2001-05-30 2002-12-05 Chiba-Prefecture Acides nucleiques isoles dans le neuroblastome
WO2002103017A1 (fr) * 2001-05-31 2002-12-27 Chiba-Prefecture Acides nucleiques isoles dans le neuroblastome
WO2004039975A1 (fr) * 2002-10-30 2004-05-13 Hisamitsu Pharmaceutical Co., Inc. Acides nucleiques isoles d'un neuroblastome au stade 4s
WO2005030959A1 (fr) * 2003-09-25 2005-04-07 Chiba-Prefecture Microreseau d'evaluation de pronostic neuroblastome et procede d'evaluation de pronostic de neuroblastome

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001066733A1 (fr) * 2000-03-07 2001-09-13 Chiba-Prefecture Sequences d'acides nucleiques presentant une expression accrue dans le neuroblastome benin par rapport au neuroblastome humain acritique
WO2001066719A1 (fr) * 2000-03-07 2001-09-13 Chiba-Prefecture Nouveau gene clone en neuroblastome humain et nouveaux fragments de gene
WO2002003071A2 (fr) * 2000-07-05 2002-01-10 Pangene Corporation Modulateurs de l'activite de la proteine trk, et compositions et methodes d'utilisation
WO2002097093A1 (fr) * 2001-05-30 2002-12-05 Chiba-Prefecture Acides nucleiques isoles dans le neuroblastome
WO2002103017A1 (fr) * 2001-05-31 2002-12-27 Chiba-Prefecture Acides nucleiques isoles dans le neuroblastome
WO2004039975A1 (fr) * 2002-10-30 2004-05-13 Hisamitsu Pharmaceutical Co., Inc. Acides nucleiques isoles d'un neuroblastome au stade 4s
WO2005030959A1 (fr) * 2003-09-25 2005-04-07 Chiba-Prefecture Microreseau d'evaluation de pronostic neuroblastome et procede d'evaluation de pronostic de neuroblastome

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
ANNEREN C. ET AL.: "The FRK7RAK-SHB signaling cascade: A versatile signal-transduction pathway that regulates cell survival differentiation and proliferation", CURRENT MOLECULAR MEDICINE, vol. 3, no. 4, 2003, pages 313 - 324, XP003020517 *
BIOCHEM. BIOPHYS. RES. COMMUN., vol. 278, no. 3, 2000, pages 537 - 543 *
CHAO M.V. ET AL.: "Neurotrophins and their receptors: A convergence point for many signalling pathways", NATURE REVIEW NEUROSCIENCE, vol. 4, 2003, pages 299 - 309, XP003020518 *
DATABASE GENPEPT [online] XP003020515, accession no. NCBI Database accession no. (NM_138356 *
FURUYA T. ET AL.: "Shinkei Gashu ni Okeru Shf (src homology domain containing F) to TrkA no Sogo Sayo (The interaction of Shf (src homology containing F) and TrkA in neuroblastoma cells)", ANNUAL MEETING OF THE JAPAN CANCER ASSOCIATION KIJI, vol. 65TH, 28 August 2006 (2006-08-28), pages 82 - 83, XP003020522 *
FURUYA T. ET AL.: "Shinkei Gashu ni Okeru Shf no Kinoteki Yakuwari ni Kansuru Kenkyu (Functional Implication of the Shf in Neuroblastoma)", JOURNAL OF NIHON UNIVERSITY MEDICAL ASSOCIATION, vol. 65, no. 6, 1 December 2006 (2006-12-01), pages 367 - 375, XP003020519 *
FURUYA T. ET AL.: "Shinkei Gashu yoru Cloning shita Shinki TrkA Ketsugo Tanpakushitsu Shf no Kino Kaiseki to Sono Shinkei Gashu ni Okeru Igi ni Tsuite", JAPANESE JOURNAL OF PEDIATRIC HEMATOLOGY, vol. 20, no. 5, 31 October 2006 (2006-10-31), pages 338, XP003020521 *
LINDHOLM C.K. ET AL.: "Shf, a Shb-like adaptor protein, is involved in PDFGF-alpha-receptor regulation of apoptosis", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 278, 2000, pages 537 - 543, XP003020516 *
LINDHOLM C.K.: "IL-2 receptor signaling through the Shb adaptor protein in T and NK cells", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 296, 2002, pages 929 - 936, XP003020524 *
MAMMALIAN GENE COLLECTION (MGC) PROGRAM TEAM: "Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences", PNAS, vol. 99, no. 26, 2002, pages 16899 - 16903, XP002331808 *
NIIZUMA H. ET AL.: "Sanpatsusei Shinkei Gashu 373 Shorei no TRK-A Hatsugen to Seizonritsu no Kaiseki", JAPANESE JOURNAL OF PEDIATRIC HEMATOLOGY, vol. 20, no. 5, 31 October 2006 (2006-10-31), pages 384, XP003020520 *
SNIDER W.D. ET AL.: "Functions of the neurotrophins during nervous system development: What the knockouts are teaching us", CELL, vol. 77, 1994, pages 627 - 638, XP003020523 *
SUGIMOTO T. ET AL.: "Signal transduction pathways through TRK-A and TRK-B receptors in human neuroblastoma cells", JPN. J. CANCER RES., vol. 92, 2001, pages 152 - 160, XP003020526 *
TSURUDA A. ET AL.: "Constitutively active Src facilitates NGF-induced phosphorylation of TrkA and causes enhancement of the MAPK signaling in SK-N-MC cells", FEBS LETTERS, vol. 560, 2004, pages 215 - 220, XP004492518 *
WOOTEN M.W. ET AL.: "Nerve growth factor stimulates multisite tyrosine phosphorylation and activation of the atypical protein kinase C's via a src kinase pathway", MOLE. CELL BIOL., vol. 21, no. 24, 2001, pages 8414 - 8427, XP003020525 *

Also Published As

Publication number Publication date
JP2008029296A (ja) 2008-02-14

Similar Documents

Publication Publication Date Title
Fukutomi et al. Hepatitis C virus core protein stimulates hepatocyte growth: Correlation with upregulation of wnt‐1 expression
Greenberg et al. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene
White et al. Genetic amplification of the transcriptional response to hypoxia as a novel means of identifying regulators of angiogenesis
EP1064404B1 (fr) Genes a regulation p53
US7459282B2 (en) Use of c-Jun or c-Jun activating agents such as UV or c-Jun N-terminal kinases (JNKs) for treating cancer
JP4276432B2 (ja) Ｍｕｃ１による細胞増殖調節方法
JP2004508031A (ja) 細胞老化関連核酸配列及びタンパク質
US20080182250A1 (en) Method for detecting multiple myeloma and method for inhibiting the same
JP2005514910A (ja) 乳癌の処置に対する遺伝子標的を同定するための方法および試薬
EP3327137B1 (fr) Facteur de régulation d'un hôte pour améliorer la prolifération et la propagation de virus de la vaccine
CN109988842B (zh) 一种寡核苷酸标记探针原位检测pdl1的试剂组与应用
JP2005504521A (ja) アクチン重合状態の調節剤を含む腫瘍病変の診断、予防又は治療用の薬剤組成物
WO2008015942A1 (fr) Agent thérapeutique contre le neuroblastome, procédé de criblage à la recherche de l'agent thérapeutique et procédé de détermination du pronostic d'un neuroblastome
CN115029438A (zh) Rarres2基因在乳腺癌脑转移诊疗中的应用
JP4122205B2 (ja) ４ｓ期神経芽細胞腫から単離された核酸
AU2018322286B2 (en) Fusion protein of DCTN1 protein with RET protein
US20190153547A1 (en) Novel cell based assay
JP2006525351A (ja) ガンを診断および治療するための方法
JPH09508006A (ja) キネシンと化学療法薬に対する感受性との関連
KR20200062721A (ko) 항암제 내성 진단 마커로서의 miR-143-3p 및 miR-373-5p, 및 이의 이용
RU2813996C2 (ru) Слитый белок из белка dctn1 с белком ret
US8852939B2 (en) Use of Vgll3 activity modulator for the modulation of adipogenesis
KR101104105B1 (ko) 대장암 진단용 조성물 및 그 용도
KR101466661B1 (ko) Ｔｍｃ５ 유전자의 신규한 용도
KR101808658B1 (ko) 암 진단 키트 및 암 예방 또는 치료용 약제학적 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07791313

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 07791313

Country of ref document: EP

Kind code of ref document: A1