WO2008066057A1

WO2008066057A1 - Control of cell proliferation targeted for iqgap3

Info

Publication number: WO2008066057A1
Application number: PCT/JP2007/072897
Authority: WO
Inventors: Sachiko Tsukita; Hisashi Nojima
Original assignee: Kyoto University
Priority date: 2006-11-30
Filing date: 2007-11-28
Publication date: 2008-06-05

Abstract

Disclosed are: a factor for controlling the expression of IQGAP3 gene or the function of IQGAP3 protein; a pharmaceutical composition comprising the factor; a pharmaceutical composition comprising IQGAP3; a vector carrying IQGAP3 gene; a cell transfected with IQGAP3 gene; a method for detection or identification of a transient amplifying (TA) cell; and others.

Description

Specification

Control of cell growth targeting IQGAP3

Technical field

[0001] The present invention relates to control of cell proliferation. Specifically, it relates to cell growth control targeting IQGAP3, which controls contact inhibition.

Background art

[0002] It is known that when cells are cultured in the in vitro port, the cells repeatedly grow until they spread over the bottom of the container (hereinafter, this state is referred to as "confluent"), and then the cell growth stops. ing. This phenomenon is called “contact inhibition of cell growth” (hereinafter abbreviated as “contact inhibition”), and is a concept established from research on fibroblasts since the 1950s. According to various studies, it is hypothesized that there is a mechanism by which cells can perceive their density rather than the growth of nutrients in the culture broth caused by changes in pH. The ability of cell-cell adhesion and its signaling pathway to be recognized to recognize cell density What is the signal-transduction pathway from cell-cell adhesion and its underlying proteins in epithelial cells and fibroblasts? Even force, even cell-to-cell adhesion, does not become a constant factor in inhibiting cell growth.

[0003] It is observed that the 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.

[0004] Further analysis of contact inhibition in more detail is also important in the development and organ formation of individuals. For example, adult hepatocytes are in the quiescent (GO) phase, with little cell division. When this liver tissue is lost due to necrosis caused by drug administration or partial excision of the liver, the remaining hepatocytes start to grow, return to their original size, and stop growing. This mechanism is still unknown. Contact inhibition may be involved in this process.

[0005] 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 literature l: Briggs, M. W., and D. B. Sacks. 2003. IQGAP proteins are integral c omponents of cycloskeletal regulation. EMBO Rep. 4: 571-574

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

Problems to be solved by the invention

[0006] 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.

Means for solving the problem

[0007] The inventors of the present invention have intensively studied to solve the above-mentioned problems, clarified that IQGAP3 is a negative regulator of contact inhibition, and prepared interfering RNA and antibodies targeting it. The present invention has been completed successfully.

[0008] That is, the present invention provides the following:

(1) Factors that suppress IQGAP3 gene expression or IQGAP3 protein function;

(2) The factor according to (1), which is selected from the group consisting of interfering RNA, antisense oligonucleotide, antibody, inhibitory peptide, and dominant negative mutant;

(3) 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;

(4) The factor according to (1), which is an antibody against IQGAP3 protein;

(5) Factors that promote the expression of IQGAP3 gene or the function of IQGAP3 protein;

(6) A pharmaceutical composition for treating a disease that is advantageous for suppressing negative regulation of cellular contact inhibition, comprising the factor according to any one of (1) to (4);

(7) The pharmaceutical composition according to (6), wherein the disease is a tumor or cancer;

(8) Negative regulation of cellular contact inhibition comprising the factor according to (5) A pharmaceutical composition for treating a disease for which it is beneficial to promote;

(9 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;

(10) a vector containing the IQGAP3 gene;

(11) cells into which IQGAP3 gene has been introduced;

(12) A cell transformed with the vector according to (10);

(13) The cell according to (11) or (12), which is a stem cell;

(14) Use of IQGAP3 gene or IQ GAP3 protein as a marker for transient amplifying (TA) cells.

(15) Treating a disease that is advantageous for suppressing negative regulation of cellular contact inhibition in a patient, characterized by administering the factor according to any one of (1) to (4) to the patient How to do;

(16) The factor according to any one of (1) to (4) for manufacturing a medicament for treating a disease for which it is advantageous to suppress negative regulation of cell contact inhibition Use of;

(17) Use of the factor according to any one of (1) to (4) above to treat a disease for which it is advantageous to suppress negative regulation of cellular contact inhibition in a subject;

(18) A method for treating a disease for which it is advantageous to promote negative regulation of cellular contact inhibition in a patient, comprising administering the factor described in (15) to the patient;

(19) Use of the factor according to (5) for the manufacture of a medicament for treating a disease for which it is advantageous to promote negative regulation of cellular contact inhibition;

(20) Use of the factor described in (5) for treating a disease for which it is advantageous to promote negative regulation of cellular contact inhibition in a subject;

(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;

(22) A kit for detecting or identifying TA cells, comprising a means for detecting IQGAP3 gene or IQGAP3 protein as a marker for TA cells. The invention's effect

[0009] According to the present invention, there are provided 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.

Brief Description of Drawings

[0010] FIG. 1 shows changes in IQGAP3 expression levels with proliferation. Figure la is the growth curve of Eph4 cells (N = 6). 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 μm. 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. As markers, 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

Yes

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, and P represents panel cells. The scale bar is 10 m.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] 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! /. For reference, the nucleotide sequence registered as XM_892303 is shown in SEQ ID NO: 1, and the amino acid sequence of IQGAP3 protein encoded thereby is shown in SEQ ID NO: 2. In addition to DNA having the nucleotide sequence set forth in SEQ ID NO: 1, 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. Here, the following conditions are exemplified as stringent hybridization conditions.

[0012] For example, using any of the following (1) to (3) as a hybridization buffer, hybridization is performed at 42 ° C:

(1) 7% SDS, 50% formamide, 5xSSC, 2% blocking reagent, 0.1% N—laurolsarcosine, 50 mM sodium phosphate, pH 7.0

(2) 50% formamide, 0.02% SDS, 5xSSC, 2% blocking reagent, 0.1% ― laurolsarcosine (3) 0.02% SDS, 5xSSC, 2% blocking reagent, 0.1% N-laurolsarcosine (Note) lxSSC (0.15M NaCl, 0.005M sodium citrate), 10% blocking reagent (Roche Blocking regent 0.1 M Maleic acid, 0.15 M NaCl, pH 7.

(It was melted so that it became 10% at 5)

Then, wash under the following conditions (1) to (3):

(1) Wash with 42 ⁰ C (trowel 1 time (2xSSC, 0.1% SDS), then wash 3 times (0. lxSSC, 0.1% SDS).

(2) 42. Wash with 1 trowel (2xSSC, 0.1% SDS), then 65. C (Wash 2 times (0. lxSSC, 0.1% SDS).

(3) 42. Wash once with a trowel (2 33 pcs, 0.1% SDS, 50% phenolemamide), then wash 2 times with C. 55 C. (0.1 lxSSC, 0.1% SDS).

[0013] The "IQGAP3 gene" of the present invention includes a human IQGAP3 gene. In the present specification, the term “IQGAP3 gene” includes DNA and RNA encoding the IQGAP3 protein.

[0014] Further, in the present specification, the term "IQGAP3 gene" includes "IQGAP3 DNA" and "IQGAP3 RNA". Therefore, the term "expression of IQGAP3 gene" includes "expression of IQGAP3 DNA" and "IQGAP3RNA". Expression ". The term “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.

[0015] In the present specification, the term "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. Further, for example, “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. Furthermore, the “IQGAP3 protein” or “IQGAP3 polypeptide” of the present invention is Also includes fragments of polypeptides with negative regulation activity of tactinhibition

In the present specification, “protein” and “polypeptide” are synonymous. “Polynucleotide” also includes DNA and RNA.

In the present specification, unless otherwise specified, “suppressing the expression” of a gene includes reducing the expression of the gene from a normal level and completely eliminating the gene expression. Specifically, `` 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. For example, the IQGAP3 gene of a cell may be knocked down, knocked out, or deleted by a known method.

In the present specification, unless otherwise specified, “suppressing the function” of a protein includes reducing the function of the protein below a normal level and completely eliminating the function.

In the present specification, unless otherwise specified, “promoting the expression” of a gene means increasing the expression of the gene from a normal level. Specifically, “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.

In the present specification, unless otherwise specified, “promoting the function” of a protein means increasing the function of the protein from a normal level.

[0021] As used herein, the term "drug" refers to a substance that has a desired therapeutic or prophylactic effect when administered!

[0022] In one embodiment, 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.

[0023] 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. In particular, interfering RNA having about 19 to about 23 bases, especially about 21 (also called siRNA) 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!

[0024] By treating the cells with these factors, for example, by contacting the cells with these factors or introducing these factors into the cells, 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. For example, 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. . In addition, 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. In addition, IQGAP3 gene expression can be suppressed or IQGAP3 protein function can be suppressed by methods other than those described above known in the art.

[0025] 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 (e.g., Yon method) has also been established and has the advantage of being easy to install and easy to use. As 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. By adopting the direct introduction method, the configuration and use of the drug delivery system and the pharmaceutical composition described below can be simplified. Conditions and factors for directly introducing the interfering RNA can be appropriately selected by those skilled in the art.

In the present invention, 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. Here, 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. Preferably, 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. In addition, these interfering RNAs may be in the form of double stranded RNA.

[0027] By using 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. Such diseases include, but are not limited to, various diseases caused by cell overgrowth, such as various tumors and cancers. Of the diseases that can be treated using a factor that suppresses the function of the IQGAP3 gene or IQGA P3 protein, a preferable disease is a tumor or a cancer, particularly a type of tumor or cancer that forms a cell mass.

[0028] Therefore, in a further aspect, 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.

[0029] Administration of the pharmaceutical composition to a patient can be performed by a known method such as oral, parenteral, or topical administration. For example, 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.

[0030] Further, 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. Provide a method of treatment.

[0031] Furthermore, 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.

[0032] In yet another embodiment, 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.

[0033] In the above pharmaceutical composition, method or use, other drugs known to inhibit cell proliferation, such as anticancer drugs (eg, adriamycin, cyclophosphamide, 5FU, cisplatin), etc. It may be combined with a factor that suppresses the function of the gene or IQGAP3 protein.

[0034] 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.

[0035] Conversely, when the function of IQGAP3 gene or IQGAP3 protein is promoted, negative regulation of cell contact inhibition is promoted, and cell proliferation is promoted. Therefore, in another embodiment, the present invention relates to a factor that promotes the function of IQGAP3 gene or IQGAP3 protein.

[0036] By treating the cells with these factors, for example, by contacting the cells with these factors or introducing these factors into the cells, 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. For example, 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 can also be contacted or introduced directly into the cell. 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. In addition, expression of IQGAP3 gene can be promoted by methods other than those described above known in the art.

[0037] 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. In addition, a factor that promotes the function of IQGAP3 gene or IQGAP3 protein may be used in regenerative medicine.

[0038] Therefore, in a further embodiment, 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.

[0039] The pharmaceutical composition can be administered to a patient by a known method such as oral, parenteral, or topical administration. For example, 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.

[0040] Further, 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.

[0041] Furthermore, 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.

[0042] In yet another embodiment, the present invention provides a cell contact inhibitor in a subject. Provided is the use of 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.

[0043] In the above pharmaceutical composition, method or use, other agents known to promote cell growth, such as growth factors (eg, EGF, PDGF, HGF, FGF), etc., are added to the IQ GAP3 gene. Or you may combine with the factor which accelerates | stimulates the function of IQGAP3 protein.

[0044] 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.

[0045] As described above, 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. In addition, IQGAP3 gene or IQGAP3 protein may be used for regenerative medicine!

[0046] 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. For example, a method using a vector (for example, a method using an adenovirus vector or the like), cell fusion, electopore position, gene gun, lipofussion, direct introduction method or the like can be used to introduce a gene into a cell. . In addition, 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. In addition, IQGAP3 gene or IQGAP3 protein can be delivered to cells by methods other than those described above known in the art.

[0047] Accordingly, 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.

[0048] The pharmaceutical composition can be administered to a patient by a known method such as oral, parenteral or topical administration. For example, 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.

[0049] Further, 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.

[0050] Furthermore, 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.

[0051] Furthermore, 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.

[0052] In the above pharmaceutical composition, method or use, other agents known to promote cell growth, such as growth factors (eg, EGF, PDGF, HGF, FGF), etc., may be used as IQ GAP3 gene Or combine with IQGAP3 protein! /.

[0053] 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. When using a vector containing the IQGAP3 gene, 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.

[0054] In a further embodiment, the present invention provides a vector in which the IQGAP3 gene is linked in an expressible state. As the backbone of the vector of the present invention, 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.

[0055] 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.

[0056] Preferably, 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. For example, 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.

[0057] Further, for example, 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.

[0058] Furthermore, 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. For detection of 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. For detection of the IQGAP3 protein, 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.

[0059] Further, the present invention provides a kit 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.

[0060] Hereinafter, the present invention will be described in more detail and specifically with reference to examples. However, the examples should not be construed as limiting the present invention.

[0061] The materials and experimental methods used in the examples will be described.

(l) cDNA cloning, sequencing and expression vectors

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).

[0062] (2) Antibody

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).

[0063] (3) Cells

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

2

The number of cells was counted. NIH 3T3 cells were cultured in a DMEM medium supplemented with 10% FCS at 37 ° C and 5% CO.

2

[0064] (4) Inhibition of IQGAP1 and 3 expression

To suppress the level of expression of IQGAP1 and 3 in Eph4 cells (originally lacking IQGAP2), 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)

[0065] The sequence of RNA generated by expressing the above vector and having the function of knocking down IQGAP1 is:

GGAUGAGUCAGCUGUGUUA (IJ number: 7) or its phase lj, certain les, GGGAAUGUAAACAAAGUC (IJ number: 8) or its phase IJ

Is an 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! / ヽ. The RNA sequence that has the function of knocking down IQGAP3 generated by expressing the above vector is:

It is an 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. These RNA sequences become double-stranded RNA with each complementary strand! /, Or may! / ヽ.

[0066] For each of IQGAPl and IQGAP3, two interfering RNA constructs (KD-1 and KD2) 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).

[0068] (6) Immunofluorescence staining and immunohistochemical staining

For immunofluorescence staining, 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.

[0069] For immunohistofluorescence staining, 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.

[0070] Light microscope (Axiophot; Carl Zeiss Microimaging, Inc.) and Plan Apochromat A 63NA 1.40 oil immersion objective lens (Carl Zeiss Microimaging, Inc.) was used to observe an appropriate combination of filter and mirror. Pictures were taken using a cool CCD camera (ORCA-ER, Hamamatsu Photonics Κ · Κ ·) controlled by Power Machintosh G5 and software package IPLab V3. 9. 3 (Scanalytics).

[0071] Experimental methods other than those described above are also known methods, or methods that can be easily performed by those skilled in the art.

Example 1

[0072] Example 1. Relationship between cell proliferation and IQGAP3 expression

The present inventors have found that IQGAP3 signal disappears after Eph4 cells (mouse epithelial cells) become confluent (data not shown). Based on this finding, the expression of cellular IQGAP3 at several cell densities was examined by immunofluorescence. To do this: 1. Place ⁴ OxlO Eph4 cells in a 12-well (well diameter 25 mm) dish (manufactured by Falcon, catalog number 353043) and 37 in DMEM + 10% FCS medium. The cells were cultured under the conditions of C and 5% CO to obtain a growth curve (Fig. La). Proliferation draws a sigmoid curve, 3-4

2

On the day, it became confluent and an epithelial sheet was formed (initial confluent state). Even after the initial confluent state was achieved, the cells repeated 2 to 3 divisions and proliferated, resulting in smaller, confluent cells. Thereafter, on the 6th day, the growth curve reached a plateau (late confluent state). The Eph4 cells on day 3 of culture were larger in size and strength than those on day 9 of culture (Fig. Lb). Next, IQGAP3 total mRNA and protein levels were examined at several time points on the growth curve of Eph4 cells. From day 0 to day 4 (until initial confluence), IQGAP3 total mRNA and protein levels increased, and then cell density increased. IQGAP3 total mRNA and protein levels decreased (Fig. lc, d).

[0073] Next, IQGAP3 distribution at several points in the cell growth curve of Eph4 cells was examined by immunofluorescence. Cells proliferated before and in the early confluence state, and IQGAP3 was abundant in most cell-cell contact areas (Figure le). Thereafter, IQGAP3 signal begins to disappear in some cells, cell density increases, and cells without IQGAP3 signal increase as cell size decreases. (Figures le and f). At least in late confluence, cell growth stopped and IQGAP3 signal was detected only in a very limited number of cells (Figure le). In this experiment, I QGAP3 was present at the contact part of the proliferating cells positive for the proliferation marker Ki-67 (Gerdes, J. et al. 1984. J. Immunology 133: 1710-1715). Is it abundant, and 100—8 for Ki-67 negative cells? The signal of 3 was not detected!

[0074] On the other hand, the same experiment revealed that IQGAP1 was concentrated at the contact portion between cells at any growth stage by immunofluorescence for IQGAPl / Ki-67 / fafadin. Fig. Lg shows the state of culture on the 9th day.

[0075] The above results were the same for salivary gland epithelial cells (CSG1) and fibroblasts (NIH3T3) in which contact inhibition occurred! (Data not shown).

[0076] From these experimental results, IQGAP3 is thought to promote cell cycle progression by negatively regulating or canceling cell-cell contact inhibition to form late-confluent cells. .

Example 2

[0077] Example 2. Action and function of IQGAP3

Next, we investigated how the effects of cells on the function of IQGAP3 were suppressed. Four Eph4 cell clones (IQGAPl—KD—1 / 2 and IQGAP3—KD—1 / 2, both of which lack IQGAP2 expression) with suppressed IQGAP3 or IQGAPl expression by expressing interfering RNA were established. did. The method was as follows. DNA oligonucleotides of the target sequence encoding two distinct intermediate parts (two for IQ GAP1 (SEQ ID NO: 3, 4), two for IQGAP3 (SEQ ID NO: 5, 6)), HI promoter interfering RNA vector (Brummelkamp, TR et al. 2002. Science 296: 550-553; Ikenouchi, J. et al. 2005. J. Cell Biol. 171: 939-945). These interfering RNA constructs were transfected into Epf4 cells, and after selection with puromycin, clones in which the expression of IQGAPl and IQGAP3 genes were reduced were selected by fluorescence staining and Western blotting.

[0078] When examined by immunoblotting, the above Eph4 clone showed IQGAPl and It was found that IQGAP3 expression was suppressed! /, (Over 97% was suppressed! /,) (Fig. 2a). Cells stably expressing scrambled sequences of KD-1 and KD-2 (Sc-1 and Sc-2) were used as controls. From the growth curves of these clones, IQGAP3 knockdown significantly delayed growth, but IQGAP1 knockdown cells (IQGAP1-KD-1 / 2) did not grow (Figure 2b). 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. On the other hand, 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). 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. Not (Figure 2d). When examined using afadin staining, the cells in the epithelial sheet knocked down IQGAP3 were more than five times the area seen from above in the late confluent state, which was larger than wild type Eph4 cells and IQGAP1 knockdown cells. (Figure 2e). On the other hand, according to electron microscope observation, the height of the cells was not significantly different between IQGAP3 knockdown cells and wild type Eph4 cells (data not shown). Therefore, the volume of IQGAP3 knockdown cells in the late confluent state is much larger than that of the wild type Eph4 crescent pack.

Example 3

Example 3. Change in cell proliferation ability when IQGAP3 is forcibly expressed

If the cultured cells are confluent and further cultured, most cells enter the G phase.

0. IQGAP3 expression vector with GFP tag on NIH3T3 fibroblasts in G phase

0

One was transfected and forced to express. Subsequently, when the cells were co-stained with 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. Example 4

[0080] Example 4. Analysis of IQGAP3 expression pattern in tissues

Adult hepatocytes are in the G phase and have little cell division. This liver set

0

If the tissue is lost due to drug-induced necrosis or partial hepatectomy, the remaining hepatocytes begin to proliferate, return to their original size and stop proliferating, but IQGAP3 is expressed in hepatocytes only during liver regeneration. It was seen. In the small intestine and large intestine, proliferation occurs in the talibut site located below the villi, and the cell group is called transit amplifying (TA) cells derived immediately after unequal division of stem cells (Booth). and Potten, 2000. J. Clin. Invest. 105: 1493-1499; Sancho et al., 2003. Curr. Opin. Cell Biol. 15: 763-770). In addition, Ki 67 signal has been reported to be associated only with TA cells.

[0081] When examined using an immunofluorescence method, a clear signal of IQGAP3 was localized in talipto in the mouse small intestine. When observed at high magnification, 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).

[0082] These results were basically common in the small and large intestines. In other words, IQGAP3 signal was also detected in Talibut Ki-67 positive TA cells in mouse colon. When observed at high magnification, there was a clear IQGAP3 signal in the cell-cell contact area of the epithelial sheet cells at the bottom of the Talibut, and they coexisted with the positive Ki-67 signal (Fig. 3b). Furthermore, IQGAP3 was localized above Musashi-1-positive stem cells in the large intestine talyp.

[0083] Thus, 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!

Example 5

Example 5. Production of antibody against IQGAP3

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.

Industrial applicability

[0085] According to the present invention, 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.

Claims

The scope of the claims

[I] A factor that suppresses IQGAP3 gene expression or IQGAP3 protein function.

[2] The factor according to claim 1, which is selected from the group consisting of interfering RNA, antisense oligonucleotide, antibody, inhibitory peptide, and dominant negative mutant.

[3] 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.

[4] The factor according to claim 1, which is an antibody against IQGAP3 protein.

[5] A factor that promotes the expression of IQGAP3 gene or the function of IQGAP3 protein.

[6] A pharmaceutical composition for treating a disease for which it is advantageous to suppress negative regulation of cellular contact inhibition, comprising the factor according to any one of claims;! To 4.

7. The pharmaceutical composition according to claim 6, wherein the disease is a tumor or cancer.

[8] A pharmaceutical composition for treating a disease for which it is advantageous to promote negative regulation of cell contact inhibition, comprising the factor according to claim 5.

[9] A pharmaceutical composition for treating a disease containing an IQGAP3 gene or IQGAP3 protein, which is advantageous for promoting negative regulation of cell contact inhibition.

[10] A vector containing the IQGAP3 gene.

[I I] Cells into which IQGAP3 gene has been introduced.

[12] A cell transformed with the vector according to claim 10.

[13] The cell according to claim 11 or 12, which is a stem cell.

[14] IQGAP3 gene or IQGAP as a marker for Transient amplifying (TA) cells

Use of 3 proteins.

[15] A disease for which it is advantageous to suppress negative regulation of cellular contact inhibition in a patient, characterized in that the factor according to any one of claims;! To 4 is administered to the patient. How to do.

[16] Use of the factor according to any one of claims;! To 4 for the manufacture of a medicament for treating a disease for which it is advantageous to suppress negative regulation of cellular contact inhibition.

[17] The use of the factor according to any one of claims 1 to 4, for treating a disease for which it is advantageous to suppress negative regulation of cellular contact inhibition in a subject.

[18] A method for treating a disease for which it is advantageous to promote negative regulation of cellular contact inhibition in a patient, comprising administering the factor of claim 5 to the patient.

[19] Use of the factor according to claim 5 for the manufacture of a medicament for treating a disease for which it is advantageous to promote negative regulation of cellular contact inhibition.

[20] Use of the factor of claim 5 for treating a disease for which it is advantageous to promote negative regulation of cellular contact inhibition in a subject.

[21] A method for detecting or identifying a Transient 讓 plifying (TA) cell, characterized by using the IQGAP3 gene or IQGAP3 protein as a marker for Transient amplifying (TA) cells.

[22] A kit for detecting or identifying TA cells, comprising a means for detecting IQGAP3 gene or IQGA P3 protein as a marker for TA cells.