[go: up one dir, main page]

WO2008066057A1 - Contrôle d'une prolifération cellulaire ciblée pour iqgap3 - Google Patents

Contrôle d'une prolifération cellulaire ciblée pour iqgap3 Download PDF

Info

Publication number
WO2008066057A1
WO2008066057A1 PCT/JP2007/072897 JP2007072897W WO2008066057A1 WO 2008066057 A1 WO2008066057 A1 WO 2008066057A1 JP 2007072897 W JP2007072897 W JP 2007072897W WO 2008066057 A1 WO2008066057 A1 WO 2008066057A1
Authority
WO
WIPO (PCT)
Prior art keywords
iqgap3
cells
gene
protein
cell
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2007/072897
Other languages
English (en)
Japanese (ja)
Inventor
Sachiko Tsukita
Hisashi Nojima
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kyoto University NUC
Original Assignee
Kyoto University NUC
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 Kyoto University NUC filed Critical Kyoto University NUC
Publication of WO2008066057A1 publication Critical patent/WO2008066057A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to control of cell proliferation. Specifically, it relates to cell growth control targeting IQGAP3, which controls contact inhibition.
  • epithelial cultured cells divide and proliferate while maintaining the sheet structure, and even if they cover the entire bottom surface of the container, they divide several times and then stop growing. Since more than 90% of cancers are derived from epithelial cells, finding a feature that suppresses the ability of epithelial cells to divide is important in considering methods for suppressing the division of cancer cells.
  • IQGAP1 A group of proteins known as the IQGAP family is known.
  • the IQGAP family is IQGAP1 It consists of 3 types of proteins (Non-patent Document 1). So far, IQGAP1 has been the most researched! /, But (Non-Patent Document 2), IQGAP2 has been researched very much! And I know nothing about IQGAP3! /, Na! /, And! /, It ’s no exaggeration! /.
  • Non-Patent Document 2 Noritake, J., T. Watanabe,. Sato, S. Wang, and. Kaibuchi. 2005.
  • IQGAP1 a key regulator of adhesion and migration. J. Cell Sci. 118: 2085-2092
  • An object of the present invention is to elucidate the function of IQGAP3 and to develop a pharmaceutical composition or the like for treating diseases by targeting IQGAP3.
  • IQGAP3 is a negative regulator of contact inhibition, and prepared interfering RNA and antibodies targeting it.
  • the present invention has been completed successfully.
  • the present invention provides the following:
  • the factor according to (1) which is selected from the group consisting of interfering RNA, antisense oligonucleotide, antibody, inhibitory peptide, and dominant negative mutant;
  • the factor according to claim 1 which is an interfering RNA comprising the nucleotide sequence shown in SEQ ID NO: 9 or 10, or an RNA having a complementary sequence thereof, or a homologous RNA having an equivalent function;
  • the factor according to (1) which is an antibody against IQGAP3 protein
  • Negative regulation of cellular contact inhibition comprising the factor according to (5)
  • a pharmaceutical composition for treating a disease comprising a QGAP3 gene or IQGAP3 protein, for which it is advantageous to promote negative regulation of cellular contact inhibition;
  • (21) A method for detecting or identifying a TA cell, characterized by using the IQGAP3 gene or IQGAP3 protein as the primary force of a TA (Transient amplifying) cell;
  • a kit for detecting or identifying TA cells comprising a means for detecting IQGAP3 gene or IQGAP3 protein as a marker for TA cells.
  • IQGAP3 gene or IQGAP3 protein IQGAP3 gene expression or factor controlling IQGAP3 protein function, a pharmaceutical composition containing them, and the like, which controls cell contact inhibition. It is also possible to treat diseases related to cell proliferation.
  • FIG. 1 shows changes in IQGAP3 expression levels with proliferation.
  • Figure lb is a thin-layer electron micrograph of Eph4 cells on day 3 and day 9 of culture. A typical cell size is indicated by a bar with an arrow. The scale bar is lO ⁇ m.
  • Figures lc and d show the mRNA levels ( Figure 1c) and protein levels (Figure 1d) of IQGAP1 and IQG AP 3 on days 1, 2, 4, 6 and 9 of culture.
  • Figure 1e is an immunofluorescent micrograph of Eph4 cells for IQGAP 3, Ki 97, and Afadin. Bars with arrows indicate the typical diameter of the cells at each time point.
  • Merge is an overlay of the IQGAP3 dyed image and the Ki-67-fafazine dyed image.
  • the scale bar is 10 ⁇ m.
  • Figure If shows the relationship between the incubation time and the area of Eph4 cells viewed from above. The area viewed from the top on the first day was taken as 1.
  • Figure lg is an immunofluorescence micrograph of Eph4 cells on day 9 of culture for IQGAPl, Ki-67, and affadin.
  • Merge is an overlay of the IQGAP1 stained image and the Ki-67-fafazine stained image.
  • the scale bar is 10 m.
  • FIG. 2 shows stable suppression of IQGAP3 in Eph4 cells.
  • Figure 2a shows the immunoblotting results.
  • C is a wild type cell (control).
  • Figure 2b shows the growth curves of Eph4 cells knocked down with IQGAP1 (IQGAP1-KD-1 / 2) and Eph4 cells knocked down with IQGAP3 (IQGAP3 KD-1 / 2).
  • Figures 2c and d show wild-type, IQGAP1 knockdown (IQGAP1-KD) and IQGAP3 knockdown (IQGAP3— on day 4 of culture (early confluent state) and day 9 of culture (late confluent state), respectively.
  • KD Cells are immunofluorescent staining images of IQGAP1 / affadin / Ki-67 and IQGAP3 / affadin / Ki-67. Bars with arrows indicate the typical diameter of the cells at each stage. The scale bar is 10 m. Merge is an overlay of IQGAP1 or 3 stained images and Ki-67-fafazine stained images.
  • Figure 2e shows culture Shown from the top of IQGAP1-KD, IQGAP3-KD, and wild-type Eph4 cells on day 4 (early confluent state) and on day 9 of culture (late confluent state). The area of wild-type Eph4 cells on day 9 of culture was defined as 1.
  • Figure 2f shows the results of forced expression of IQGAP3 in late confluent NIH-3T3 cells.
  • GFP—IQGAP3, Ki—67 and D API were used.
  • Merge is an overlay of GFP—IQGAP3 'DAPI image and Ki 67 image.
  • the scale bar is 10 ⁇ m
  • FIG. 3 is an immunofluorescence-stained image showing the location of IQGAP3 in the small intestine (a) and large intestine (b).
  • V represents villi
  • Cr represents talipto
  • P represents panel cells.
  • the scale bar is 10 m.
  • the target molecule of the present invention is an IQGAP3 gene or IQGAP3 protein.
  • the cDNA encoding the full-length mouse I QGAP3 protein is registered with GenBank / EMBL / DDBJ under the accession number XM-892303! /.
  • the nucleotide sequence registered as XM_892303 is shown in SEQ ID NO: 1
  • the amino acid sequence of IQGAP3 protein encoded thereby is shown in SEQ ID NO: 2.
  • DNA having nucleotide sequence identity of 70% or more, preferably 80% or more, more preferably 85% or more is also included in the “IQGAP3” of the present invention. Included in “gene”.
  • the DNA having the nucleotide sequence set forth in SEQ ID NO: 1 is also included in the “IQ GAP3 gene” of the present invention, which hybridizes under stringent hybridization conditions! Is done.
  • the following conditions are exemplified as stringent hybridization conditions.
  • hybridization is performed at 42 ° C:
  • the "IQGAP3 gene” of the present invention includes a human IQGAP3 gene.
  • the term “IQGAP3 gene” includes DNA and RNA encoding the IQGAP3 protein.
  • IQGAP3 gene includes “IQGAP3 DNA” and “IQGAP3 RNA”. Therefore, the term “expression of IQGAP3 gene” includes “expression of IQGAP3 DNA” and “IQGAP3RNA”. Expression ".
  • gene or “oligonucleotide” includes both DNA and RNA. In the present specification, the nucleotide sequence notation is from 5 ′ to 3 ′ from left to right unless otherwise specified.
  • IQGAP3 protein or “IQGAP3 polypeptide” refers to a protein encoded by the above-mentioned "IQGAP3 gene", which has a negative regulation activity of contact inhibition. Everything is included.
  • the “IQGAP3 protein” of the present invention includes human IQGAP3 protein.
  • “IQGAP3 protein” refers to, for example, 1 to about 490 amino acid sequence of IQGAP3 protein encoded by DNA having the nucleotide sequence of SEQ ID NO: 1, preferably 1 to about 330, Preferably, it may be a protein having 1 to about 250 amino acid strength substitutions, insertions, additions or deletions.
  • the “IQGAP3 protein” or “IQGAP3 polypeptide” of the present invention is Also includes fragments of polypeptides with negative regulation activity of tactinhibition
  • protein and “polypeptide” are synonymous.
  • Polynucleotide also includes DNA and RNA.
  • suppressing the expression” of a gene includes reducing the expression of the gene from a normal level and completely eliminating the gene expression.
  • ⁇ suppressing expression '' of IQGAP3 gene includes inactivating or eliminating IQGAP3 gene synthesis, shifting IQGAP3 gene control 'regulatory mechanism in the direction of expression suppression, etc. To do.
  • Various means and methods for suppressing gene expression are known to those skilled in the art.
  • the IQGAP3 gene of a cell may be knocked down, knocked out, or deleted by a known method.
  • “suppressing the function” of a protein includes reducing the function of the protein below a normal level and completely eliminating the function.
  • promoting the expression” of a gene means increasing the expression of the gene from a normal level.
  • “promoting expression” of IQGAP3 gene includes activating synthesis of IQGAP3 gene, shifting IQGAP3 gene control ′ regulatory mechanism in the direction of promoting expression, and the like.
  • Various means and methods for promoting gene expression are known to those skilled in the art. Examples thereof include incorporating a promoter derived from a heterologous organism upstream of the target gene in the expression vector.
  • promoting the function” of a protein means increasing the function of the protein from a normal level.
  • drug refers to a substance that has a desired therapeutic or prophylactic effect when administered!
  • the present invention relates to a factor that suppresses the function of IQGAP3 gene or IQGAP3 protein. Suppressing IQGAP3 gene or IQGAP3 protein function suppresses negative regulation of cellular contact inhibition and Proliferation is suppressed.
  • Factors that suppress the function of the IQGAP3 gene or IQGAP3 protein include factors and drugs such as interfering RNA, antisense oligonucleotides, ribozymes, antibodies, abutama, inhibitory peptides, and dominant negative mutants. It is not restricted to these.
  • interfering RNA having about 19 to about 23 bases, especially about 21 is particularly preferred because it has high knockdown efficiency and is easy to prepare and handle.
  • the interfering RNA of the present invention against IQGAP3 DNA may target IQGAP3 DNA! /, As long as the effect is obtained!
  • the expression of the IQG AP3 gene in the cells at the desired site is suppressed. be able to.
  • Cells may be treated with genes encoding these factors.
  • Methods for treating cells with these factors and genes for example, methods for bringing these factors or genes encoding these factors into contact with cells or introducing them into cells are known in the art.
  • a vector-based method for example, a method using an adenovirus vector, etc.
  • cell fusion electopore position, gene gun, lipofussion, direct introduction method, etc. can be used to introduce a gene into a cell. .
  • these factors may be directly contacted or introduced into cells.
  • a person skilled in the art can appropriately select and use these methods according to the type of cells at the desired site, the type of factors to be used, and the like.
  • IQGAP3 gene expression can be suppressed or IQGAP3 protein function can be suppressed by methods other than those described above known in the art.
  • Examples of preferable factors that suppress the expression of IQGAP3 gene used in the present invention include interfering RNA for IQGAP3, antisense oligonucleotide for IQGAP3 gene, and the like.
  • Interfering RNA against IQGAP3 Antisense oligonucleotides against RNA and IQGAP3 gene have the advantage that the specificity to IQGAP3 gene is high or the risk of side effects is high with high knockout efficiency.
  • Methods for producing antisense oligonucleotides and interfering RNAs, and preferred cathodic IJs are also known to those skilled in the art and can be easily obtained.
  • Methods for introducing antisense oligonucleotides or interfering RNA into cells have also been established and has the advantage of being easy to install and easy to use.
  • a method for introducing the interfering RNA of the present invention into the body a known method such as a direct introduction method, a method using an adenovirus vector, a transdermal lipofussion or an electoral position can be used.
  • a preferred method for introducing the interfering RNA of the present invention into the body is a direct introduction method.
  • a preferred interfering RNA against IQGAP3 is a force comprising the nucleotide sequence shown in SEQ ID NO: 9 or 10 or a complementary sequence thereof, or their homologue RNA.
  • the preferred length of these interfering RNAs is about 19 to about 23 bases, and the more preferred length is about 21 bases.
  • the homologous RNA includes all RNAs having an IQGAP3 gene expression inhibitory effect equivalent to that of the interfering RNA containing the nucleotide sequence shown in SEQ ID NO: 9 or 10.
  • the interfering RNA homologue RNA of the present invention is a nucleotide sequence of interfering RNA comprising the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10, or a complementary sequence thereof, wherein 1 to several nucleotides are deleted, It has a substituted and / or added sequence (which may be a combination thereof).
  • the term “several” means usually 2 to 5, preferably 2 to 4, more preferably 2 to 3, and most preferably 2.
  • these interfering RNAs may be in the form of double stranded RNA.
  • a factor that suppresses the function of the IQGAP3 gene or IQGAP3 protein it is possible to treat a disease for which it is advantageous to suppress negative regulation of contact inhibition.
  • diseases include, but are not limited to, various diseases caused by cell overgrowth, such as various tumors and cancers.
  • a preferable disease is a tumor or a cancer, particularly a type of tumor or cancer that forms a cell mass.
  • the present invention relates to a disease in which it is advantageous to suppress negative regulation of cellular contact inhibition, including a factor that suppresses the function of IQGAP3 gene or IQGAP3 protein as described above.
  • a pharmaceutical composition for treating To do To do.
  • Administration of the pharmaceutical composition to a patient can be performed by a known method such as oral, parenteral, or topical administration.
  • parenteral administration such as injection, infusion, transdermal administration, suppository administration, etc. may be used.
  • the dosage form may be a solid form such as powder, granule, tablet, capsule or the like, or a liquid form, pasta form or ointment form.
  • the present invention provides a disease that is advantageous for suppressing negative regulation of contact inhibition in a patient, characterized in that a factor that suppresses IQGAP3 gene or IQGAP3 protein function is administered to the patient.
  • a factor that suppresses IQGAP3 gene or IQGAP3 protein function is administered to the patient.
  • the present invention suppresses the function of IQGAP3 gene or IQGAP3 protein in the manufacture of a pharmaceutical composition for treating a disease for which it is advantageous to suppress negative regulation of contact inhibition in a patient. Provide the use of factors.
  • the present invention suppresses the function of IQGAP3 gene or IQGAP3 protein for treating a disease for which it is advantageous to suppress negative regulation of cellular contact inhibition in a subject. Provide the use of factors.
  • anticancer drugs eg, adriamycin, cyclophosphamide, 5FU, cisplatin
  • a factor that suppresses the function of the gene or IQGAP3 protein may be combined with a factor that suppresses the function of the gene or IQGAP3 protein.
  • the dose of a factor that suppresses IQGAP3 gene or IQGAP3 protein function to the patient depends on the type and severity of the patient's symptoms, the patient's health condition, the treatment the patient is receiving, the patient's age, sex, This is the ability of the attending physician or specialist to make decisions according to normal procedures, taking weight and other factors into account.
  • the present invention relates to a factor that promotes the function of IQGAP3 gene or IQGAP3 protein.
  • IQG in the cells at the desired site can be obtained. It can promote the expression of AP3 gene.
  • Cells may be treated with genes encoding these factors.
  • Methods for treating cells with these factors and genes for example, methods for bringing these factors or genes encoding these factors into contact with cells or introducing them into cells are known in the art.
  • a vector-based method for example, a method using an adenovirus vector, etc.
  • cell fusion electopore position
  • gene gun lipofussion
  • direct introduction method etc.
  • IQGAP3 gene can be promoted by methods other than those described above known in the art.
  • a factor that promotes the function of IQGAP3 gene or IQGAP3 protein can be used to treat a disease for which it is advantageous to promote negative regulation of contact inhibition.
  • a factor that promotes the function of IQGAP3 gene or IQGAP3 protein may be used in regenerative medicine.
  • the present invention treats a disease that is advantageous for promoting cell contact inhibition, comprising a factor that promotes the function of IQGAP3 gene or IQGAP3 protein as described above.
  • a pharmaceutical composition is provided.
  • the pharmaceutical composition can be administered to a patient by a known method such as oral, parenteral, or topical administration.
  • parenteral administration such as injection, infusion, transdermal administration, suppository administration, etc. may be used.
  • the dosage form may be a solid form such as powder, granule, tablet, capsule or the like, or a liquid form, pasta form or ointment form.
  • the present invention provides a disease advantageous for promoting negative regulation of contact inhibition in a patient, characterized by administering to the patient a factor that promotes IQGAP3 gene or IQGAP3 protein function. Provide a method of treatment.
  • the present invention promotes the function of IQGAP3 gene or IQGAP3 protein in the manufacture of a pharmaceutical composition for treating a disease for which it is advantageous to promote negative regulation of contact inhibition in a patient. Provide the use of factors.
  • the present invention provides a cell contact inhibitor in a subject.
  • a factor that promotes the function of IQGAP 3 gene or IQGAP3 protein to treat a disease for which it is advantageous to promote Yong's negative regulation.
  • IQGAP3 gene or IQGAP3 protein-enhancing factor doses to patients depend on the type and severity of the patient's symptoms, the patient's health, the treatment they are receiving, the patient's age, gender, This is the ability of the attending physician or specialist to make decisions according to normal procedures, taking weight and other factors into account.
  • IQGAP3 protein has a negative regulatory activity of cell contact inhibition. Therefore, using the IQGAP3 gene or IQGAP3 protein, it is possible to treat diseases for which it is advantageous to promote the negative regulation activity of cell contact inhibition. Such diseases are as described above.
  • IQGAP3 gene or IQGAP3 protein may be used for regenerative medicine!
  • IQGAP3 By treating the cells with the IQGAP3 gene or IQGAP3 protein, for example, by bringing the cells into contact with these genes or proteins or introducing these genes or proteins into the cells, IQGAP3 It can promote gene expression or increase the amount of IQGAP3 protein.
  • Methods for treating cells with these genes or proteins for example, methods for bringing these factors or genes encoding these factors into contact with cells or introducing them into cells are known in the art.
  • a method using a vector for example, a method using an adenovirus vector or the like
  • cell fusion for example, electopore position, gene gun, lipofussion, direct introduction method or the like can be used to introduce a gene into a cell. .
  • IQGAP3 protein may be directly contacted or introduced into cells. Those skilled in the art can appropriately select and use these methods according to the type of cells at the desired site, the type of factors used, and the like.
  • IQGAP3 gene or IQGAP3 protein can be delivered to cells by methods other than those described above known in the art.
  • the present invention in a further aspect, provides an IQGAP3 gene or IQGAP3 protein. It is intended to provide a pharmaceutical composition for treating diseases in which it is beneficial to promote negative regulation of cellular contact inhibition, including white.
  • the pharmaceutical composition can be administered to a patient by a known method such as oral, parenteral or topical administration.
  • parenteral administration such as injection, infusion, transdermal administration, suppository administration, etc. may be used.
  • the dosage form may be a solid form such as powder, granule, tablet, capsule or the like, or a liquid form, pasta form or ointment form.
  • the present invention provides a method for treating a disease that is advantageous in promoting negative regulation of cellular contact inhibition in a patient, characterized by administering the IQGAP3 gene or IQGAP3 protein to the patient. provide.
  • the present invention relates to the use of IQG AP3 gene or IQGAP3 protein in the manufacture of a pharmaceutical composition for treating a disease for which it is advantageous to promote negative regulation of cellular contact inhibition in a patient. provide.
  • the present invention provides use of IQGAP3 gene or IQGAP3 protein for treating a disease for which it is advantageous to promote negative regulation of cellular contact inhibition in a patient.
  • IQ GAP3 gene may be used as IQ GAP3 gene Or combine with IQGAP3 protein! /.
  • growth factors eg, EGF, PDGF, HGF, FGF
  • the dose of IQGAP3 gene or IQGAP3 protein to a patient depends on various factors such as the type and severity of the patient's symptoms, the patient's health, the treatment the patient is receiving, the patient's age, sex, and weight.
  • the attending physician or specialist can determine the dosage according to normal procedures.
  • the efficiency with which the vector is introduced into the cell and the expression efficiency of the introduced vector vary depending on the type of vector and the type of cell.
  • the amount of vector to be determined can be determined.
  • the present invention provides a vector in which the IQGAP3 gene is linked in an expressible state.
  • a vector in which the IQGAP3 gene is linked in an expressible state for example, known vectors such as pcDNA3 and pCAGGS can be used. Replication start point, Enhancer array Mouth motor arrangements and the like are known to those skilled in the art.
  • the IQGAP3 gene of the present invention can be transformed into appropriate cells using methods known to those skilled in the art to produce IQGAP3 protein.
  • the above-described vector of the present invention may be used. Transformation methods are known to those skilled in the art. For example, a method using a vector (for example, a method using an adenovirus vector), a cell fusion, an electopore position, a gene gun, a lipofussion, a direct introduction method or the like should be used. Can do. A person skilled in the art can select and use these methods as appropriate according to the type of cells at the desired site, the type of factors used, and the like.
  • the cell into which the IQGAP3 gene is introduced is a human cell, and more preferably a patient-derived cell that requires enhanced action of the IQGAP3 protein.
  • cells can be collected from a site that requires enhanced action of IQGAP3 protein in a patient, and the collected cells can be transformed with a vector containing the IQGAP3 gene of the present invention. If desired, the transformed cells are cultured and expanded, and the transformed cells are returned to the patient, thereby enhancing the action of the IQGAP3 protein in the patient.
  • IQGAP3 gene may be introduced into the stem cell by a known method after the stem cell is isolated. Stem cells into which the IQGAP3 gene has been introduced may cause unequal division activation, increasing their usefulness.
  • the present invention provides a method for detecting or identifying TA cells, which comprises using IQGAP3 gene or IQGAP3 protein as a marker for TA (Transient Amplifying) cells.
  • IQGAP3 gene expression an oligonucleotide that is a hybridizing oligonucleotide and bound to a detectable label may be used as a probe. If amplification of the IQGAP3 gene is required, a known gene amplification method such as RT-PCR may be used.
  • an antibody that specifically binds to this and to which a detectable label is bound may be used.
  • Those skilled in the art can easily determine and select the sequence and production method of oligonucleotide probes for detecting IQGAP3 gene expression, primers necessary for gene amplification, and antibodies for detecting IQGAP3 protein. Can do.
  • kits for detecting or identifying TA cells comprising IQGAP3
  • a kit comprising means for detecting a gene or IQGAP3 protein as a marker for TA (Transient amplifying) cells is provided.
  • the kit of the present invention includes an oligonucleotide probe that is a hybridization oligonucleotide to the IQGAP3 gene and bound to a detectable label, or means for amplification of the IQGAP3 gene, such as primers for RT-PCR. May be.
  • the kit of the present invention may include IQGAP3, an antibody that specifically binds to a protein and has a detectable label, or a means for staining.
  • CDNA encoding the entire ORGA of IQGAP1, 2 and 3 was amplified from a protein coding region library of mouse small intestine cDNA.
  • the amplified cDNA was subcloned into pGEM-T Easy Vect or (Promega), and the nucleic acid sequence was determined from each amplified fragment.
  • Sequence data for cDNAs encoding full-length mouse IQGAP1, 2 and 3 are available from GenBank / EMBL / DDBJ (accession numbers AF240630, AK147360, and XM-892303, respectively).
  • Anti-mouse IQGAP1, 2 and 3 polyclonal antibodies were raised in rabbits against GST fusion proteins containing amino acids at positions 257-523, 401-668, and 185-581 of the amino terminal domain, respectively.
  • Mouse anti-IQGAP1 monoclonal antibody was purchased from Transduction Laboratories (San Diego, Calif.).
  • Rat anti-IQGAP3 monoclonal antibody was obtained as follows. Wistar rats were immunized with a GST fusion protein containing amino acids at positions 185-581 of the N-terminal domain of IQGAP3, and lymphocytes were fused with P3 myeloma cells to obtain hyperidoma cells. All antibodies reacted specifically with antigen in immunoblotting and immunofluorescence staining.
  • Anti-fafazine antibody (Saki saka, T. et al "1999. Oncogene 18: 1609-1617), anti-Musashi-1 antibody (Kaneko, Y. et al ,. 2000. Dev. Neurosci. 22: 139-153) Dr. Takai (Osaka University graduate School of Medicine) graduate school) and Dr. Okano (Keio University School of Medicine).
  • Eph4 Mouse mammary epithelial cells Eph4 were cultured in DMEM medium supplemented with 10% FCS at 37 ° C. and 5% CO. Cell counts can be obtained by plating 4 lxlO on a 12 wel plate
  • NIH 3T3 cells were cultured in a DMEM medium supplemented with 10% FCS at 37 ° C and 5% CO.
  • a DNA oligonucleotide of the target sequence that encodes two different intermediates was transferred to the HI promoter interfering RNA vector (Brummelkamp, TR et al ,. 2002. Scien ce 296: 550-553; Ikenouchi, J. et al., 2005. J. Cell Biol. 171: 939-945) ligated downstream of the HI promoter. (The part that functions as a lowercase letter indicates the connected part):
  • IQGAP1 D 1 GGATGAGTCAGCTGTGTTAttcaagagaTAACACAGCTGACTCAT CC (SEQ ID NO: 3)
  • IQGAP1 D2 GGGAATGTAAACAAAGTCAttcaagagaTGACTTTGTTTACATTC CC (SEQ ID NO: 4)
  • IQGAP3 KD 1 GCTGTGTGGAGGATCAACAttcaagagaTGTTGATCCTCCACACA GC (SEQ ID NO: 5)
  • IQGAP3 D2 GTCAGCCGTGGTTCTGATTttcaagagaAATCAGAACCACGGCTG AC (SEQ ID NO: 6)
  • RNA generated by expressing the above vector and having the function of knocking down IQGAP1 is:
  • RNA sequence comprising The preferred length of these RNA sequences is about 19 to about 23 bases, and the more preferred length is about 21 bases. These RNA sequences become double-stranded RNA with each complementary strand! /, Or may! / ⁇ .
  • the RNA sequence that has the function of knocking down IQGAP3 generated by expressing the above vector is:
  • RNA sequence containing GCUGUGUGGAGGAUCAACA (IJ number: 9) or its phase lj, or GUCAGCCGUGGUUCUGAUU (IJ number: 10) or its phase IJ.
  • the preferred length of these RNA sequences is about 19 to about 23 bases, and the more preferred length is about 21 bases.
  • RNA constructs were transfected into Eph4 cells to suppress the expression of IQGAPl and IQGAP3. After selection with puromycin, clones with reduced expression of each gene were selected by fluorescent staining and Western blot. Four or more clones with decreased expression of each target gene were screened and used in the experiment. Proteins were separated by one-dimensional SDS-PAGE, transferred from the gel onto a nitrocellulose membrane, and then incubated with a primary antibody against the protein of interest. The bound antibody was detected with an HRP-conjugated secondary antibody (GE Healthcare).
  • HRP-conjugated secondary antibody HRP-conjugated secondary antibody
  • cells were fixed with 1% formalin, treated with 0.1% Triton X 100 in PBS, then blocked with 1% BSA, reacted with the primary antibody against the target protein, PBS Then, it was reacted with a secondary antibody, washed with PBS, embedded in an aqueous sealant, and observed with a fluorescence microscope.
  • the tissue was embedded in Tissue—Tek, cut to a thickness of 7 m with a cryostat, the sample was fixed with 1% formalin, and 0.1% Triton X in PBS. — Treated with 100, blocked with 1% BSA, reacted with primary antibody against target protein, washed with PBS, reacted with secondary antibody, washed with PBS, embedded in aqueous mounting medium Then, it observed with the fluorescence microscope.
  • Example 1 Relationship between cell proliferation and IQGAP3 expression
  • IQGAP3 is thought to promote cell cycle progression by negatively regulating or canceling cell-cell contact inhibition to form late-confluent cells. .
  • IQGAP3—KD—1 / 2 Cells that had IQGAP3 knocked down (IQGAP3—KD—1 / 2) had a growth rate of about 1/3 that of the target wild type cells before confluence and during confluence.
  • the cells in which IQG AP 1 was knocked down showed the same behavior as the wild type.
  • IQGAP3 and IQGAP1 signals were not observed at the cell-cell contact sites in both IQGAP3 and IQGAP1 knockdown cells before confluence and during confluence (Fig. 2c). , d).
  • d In late confluence, there was a significant size difference between wild-type and IQGAP3 knockdown cells, but there was a significant size difference between wild-type and IQGAP1 knockdown cells.
  • Ki-67 a marker for cell proliferation
  • Ki-67 was also positive in cells expressing GFP- IQGAP3 (Fig. 2f). This indicates that the cell cycle begins to rotate when IQGAP3 is forcibly expressed.
  • TA transit amplifying
  • a clear signal of IQGAP3 was localized in talipto in the mouse small intestine.
  • the IQGAP3 signal was confined to the cell-cell contact area of Ki-67 positive TA cells! /, (Fig. 3a).
  • the small intestine was immunostained in triplicate for IQGA P3, DAPI and Musashi-1 (stem cell marker; Kaneno et al., 2000. Dev. Neuros ci. 22: 139-153).
  • a clear IQGAP3 signal was localized at the cell contact site! /, (Fig. 3a).
  • a faint dispersion of IQGAP3 signal is detected in Musashi-1 positive stem cells, which may be the force present above the panel cell compartment or mixed with the panel cells at the bottom of the script (Kayahara et al. ., 20 03. FEBS Lett. 535: 131-135).
  • IQGAP3 expression was specific to TA cells. Therefore, IQGAP3 gene or IQGAP3 protein can be used as a marker that can label TA cells.
  • TA The cells are very interesting as research subjects because they combine their strong ability to proliferate and progenitor cells into the intestinal epithelium!
  • An anti-mouse IQGAP3 polyclonal antibody was obtained in rabbits using a GST fusion protein containing amino acids 185 to 581 of the amino terminal domain of mouse IQGAP3.
  • a rat anti-IGGAP3 monoclonal antibody was obtained as follows. Using a GST fusion protein containing amino acids 185 to 581 of the amino terminal domain of mouse IQGAP3, immunize the Wister rat, obtain lymphocytes, and fuse with P3 myeloma cells to obtain high-pridoma cells. It was. The antibody thus obtained specifically reacted with IQG AP3 in immunofluorescence staining and immunoplotting.
  • IQGAP3 gene or IQGAP3 protein, IQGAP3 gene expression or factor controlling IQGAP3 protein function, a pharmaceutical composition containing them, and the like are provided to control cell contact inhibition. And treatment of diseases related to cell proliferation is provided. Therefore, the present invention can be used in the field of pharmaceuticals, research reagents such as pathology, and test drugs.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Analytical Chemistry (AREA)
  • Public Health (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne : un facteur pour contrôler l'expression du gène IQGAP3 ou la fonction d'une protéine IQGAP3 ; une composition pharmaceutique comprenant le facteur ; une composition pharmaceutique comprenant IQGAP3 ; un vecteur portant le gène IQGAP3 ; une cellule transfectée par le gène IQGAP3 ; un procédé de détection ou d'identification d'une cellule d'amplification transitoire (TA) ; et autres.
PCT/JP2007/072897 2006-11-30 2007-11-28 Contrôle d'une prolifération cellulaire ciblée pour iqgap3 Ceased WO2008066057A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2006324211 2006-11-30
JP2006-324211 2006-11-30

Publications (1)

Publication Number Publication Date
WO2008066057A1 true WO2008066057A1 (fr) 2008-06-05

Family

ID=39467845

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2007/072897 Ceased WO2008066057A1 (fr) 2006-11-30 2007-11-28 Contrôle d'une prolifération cellulaire ciblée pour iqgap3

Country Status (1)

Country Link
WO (1) WO2008066057A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011031482A3 (fr) * 2009-08-25 2011-08-04 Opko Curna, Llc Traitement de maladies associées à la protéine d'activation de gtpase contenant un motif iq (iqgap), par inhibition d'un transcrit antisens naturel de iqgap
CN118048456A (zh) * 2024-01-30 2024-05-17 中山大学 一种作为肝癌微血管侵犯中诊断、治疗和预后的生物标志物及其应用
CN119185512A (zh) * 2024-08-29 2024-12-27 南京医科大学 Iqgap3在制备防治动脉粥样硬化药物中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006512923A (ja) * 2002-10-18 2006-04-20 エルジー ライフサイエンス リミテッド 癌関連遺伝子ファミリー

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006512923A (ja) * 2002-10-18 2006-04-20 エルジー ライフサイエンス リミテッド 癌関連遺伝子ファミリー

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 27 April 2006 (2006-04-27), "PREDICTED: Mus musculus IQ motif containing GTPase activating protein 3, transcript variant 1 (Iqgap3), mrna", Database accession no. (XM_892303) *
FURUKAWA Y. ET AL.: "Identification and characterization of a novel member of IQGAP family protein involved in the motility and invasion of cancer cells", PROCEEDINGS OF THE JAPANESE CANCER ASSOCIATION, vol. 65TH, August 2006 (2006-08-01), pages 455 *
JINAWATH N. ET AL.: "IQGAP3 promotes tumor cell migration and invasion via interactions with Cdc42, Rac1, RhoA, and R-Ras, and its involved in cytokinesis", PROC. AMER ASSOC. CANCER RES. MEETING ABSTRACTS, vol. 47, April 2006 (2006-04-01), pages 429 *
NOJIMA H. ET AL.: "The merlin tumor suppressor localization and binding partner", ANNUAL MEETING OF THE MOLECULAR BIOLOGY SOCIETY OF JAPAN, vol. 28TH, 2005, pages 271 *
WANG S. ET AL.: "IQGAP3 is a novel effector or Rac1 and Cdc42, and regulates neurite outgrowth", BULLETIN OF THE JAPANESE SOCIETY FOR NEUROCHEMISTRY, vol. 45, no. 2-3, August 2006 (2006-08-01), pages 427 *
WANG S. ET AL.: "IQGAP3, a novel effector of Rac1 and Cdc42, regulates neurite outgrowth", J. CELL SCI., vol. 120, February 2007 (2007-02-01), pages 567 - 577, XP002678880, DOI: doi:10.1242/jcs.03356 *
WANG S. ET AL.: "IQGAP3, a novel effector or Rac1, and Cdc42, regulates neurite outgrow through actin cytoskeletal reorganization", BULLETIN OF THE JAPANESE SOCIETY FOR NEUROCHEMISTRY, vol. 44, no. 2-3, August 2005 (2005-08-01), pages 184 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011031482A3 (fr) * 2009-08-25 2011-08-04 Opko Curna, Llc Traitement de maladies associées à la protéine d'activation de gtpase contenant un motif iq (iqgap), par inhibition d'un transcrit antisens naturel de iqgap
US9023822B2 (en) 2009-08-25 2015-05-05 Curna, Inc. Treatment of 'IQ motif containing GTPase activating protein' (IQGAP) related diseases by inhibition of natural antisense transcript to IQGAP
US9528110B2 (en) 2009-08-25 2016-12-27 Curna, Inc. Treatment of ‘IQ motif containing gtpase activating protein’ (IQGAP) related diseases by inhibition of natural antisense transcript to IQGAP
CN118048456A (zh) * 2024-01-30 2024-05-17 中山大学 一种作为肝癌微血管侵犯中诊断、治疗和预后的生物标志物及其应用
CN118048456B (zh) * 2024-01-30 2025-02-14 中山大学 一种作为肝癌微血管侵犯中诊断、治疗和预后的生物标志物及其应用
CN119185512A (zh) * 2024-08-29 2024-12-27 南京医科大学 Iqgap3在制备防治动脉粥样硬化药物中的应用
CN119185512B (zh) * 2024-08-29 2025-07-25 南京医科大学 Iqgap3在制备防治动脉粥样硬化药物中的应用

Similar Documents

Publication Publication Date Title
CN109797151B (zh) Circ-CDH1抑制剂的应用
Nakatsukasa et al. Tumor-associated calcium signal transducer 2 is required for the proper subcellular localization of claudin 1 and 7: implications in the pathogenesis of gelatinous drop-like corneal dystrophy
JP3730623B2 (ja) ヒト子宮頸部癌２癌原遺伝子およびそれによってコードされるタンパク質
KR20010086070A (ko) 혈관신생 및 심혈관형성의 촉진 또는 억제
Ozbun et al. Identification of differentially expressed nucleolar TGF-β1 target (DENTT) in human lung cancer cells that is a new member of the TSPY/SET/NAP-1 superfamily
TW200920406A (en) EBI3, DLX5, NPTX1 and CDKN3 for target genes of lung cancer therapy and diagnosis
Pradella et al. A ligand-insensitive UNC5B splicing isoform regulates angiogenesis by promoting apoptosis
CN102272157B (zh) 用于抑制cripto/grp78复合物形成和信号的组合物和方法
US12421514B2 (en) Methods for diagnosing and treating metastatic cancer
Lin et al. Small cytoskeleton-associated molecule, fibroblast growth factor receptor 1 oncogene partner 2/wound inducible transcript-3.0 (FGFR1OP2/wit3. 0), facilitates fibroblast-driven wound closure
WO2004006898A2 (fr) Utilisation de composes pouvant inhiber le traitement proteolytique de semaphorines pour la prevention, le traitement, le diagnostic et le pronostic d'une maladie invasive
WO2008066057A1 (fr) Contrôle d'une prolifération cellulaire ciblée pour iqgap3
JP3428441B2 (ja) タイトジャンクション構成膜蛋白質クローディンファミリー
US9198966B2 (en) B-type plexin antagonists and uses thereof
CN101952433B (zh) 在疾病的诊断与治疗中的PTHrP，其亚型及其拮抗剂
JP3288716B2 (ja) ヒト接着分子オクルディン
Saito et al. Frequent association of alternative splicing of NER, a nuclear hormone receptor gene in cancer tissues
JPWO2005111213A1 (ja) Ｍｙｃ標的遺伝子ｍｉｍｉｔｉｎ
CN110563830A (zh) Anxa1衍生多肽及其应用
CA2773614A1 (fr) Procede de traitement du cancer par inhibition de l'expression ou l'activite de trim59
EP2169060B1 (fr) Nouveau polypeptide utile pour diagnostiquer et pour traiter le cancer
KR101808658B1 (ko) 암 진단 키트 및 암 예방 또는 치료용 약제학적 조성물
US20040209809A1 (en) Shc modulation and uses thereof
CN101161283A (zh) CMTM1-v17的新应用及其拮抗剂
JPWO2005061704A1 (ja) 癌の予防・治療剤

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: 07832619

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07832619

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP