JP4510490B2 - Method for controlling cell proliferation using Jab1 / COP9 signalosome - Google Patents

Description

  The present invention relates to a method for controlling cell proliferation and the like using Jab1 / COP9 signalosome. The present invention can be used for various purposes for the purpose of suppressing or promoting cell proliferation, and can also be used as a screening method for anticancer agents and anti-inflammatory agents.

  As shown in FIG. 17, COP9 signalosome (CSN) converts various signals inside and outside the cell into intracellular signals and induces various cellular responses via various functional molecules downstream thereof. Mechanism. The present inventor has focused on Jab1 (CSN5), which is the fifth subunit, and as a result of research and analysis, (1) Jab1 has promoted the degradation of Cdk inhibitory protein p27, and (2) Jab1. Overexpression of the gene in mammalian fibroblasts reduces serum dependency in cell growth, (3) In pancreatic cancer cells, strong expression of Jab1 protein is observed in both cytoplasm and nucleus, etc. (See Non-Patent Documents 1 and 2 below).

Nature, Vol.398, No.6723, pp.160-165, 11 March 1999 Oncology Reports 11: 277-284, 2004

  The inventor pays attention to the above-mentioned functions and actions of COP9 signalosome and its constituent Jab1, and can change various cell responses such as cell proliferation by changing its activity or expression. I thought. The COP9 signalosome receives various signals and can control downstream functional molecule groups in a coordinated manner. Therefore, if cell growth and the like can be controlled by such a method, the control method includes (1) target cell. It is considered that there are advantages such as that it is not necessary to prepare a number of signal factors according to (2), and that functional molecules can be controlled collectively.

  The present invention has been made in view of the above circumstances, and its purpose is to control cell proliferation by controlling the expression or activity of Jab1 / COP9 signalosome using a control method such as cell proliferation, particularly Jab1. It is to provide a method and the like.

  As a result of conducting extensive research in view of the above problems, the present inventor (1) suppressed the intracellular expression of Jab1 gene by RNAi method for various human-derived cancer cells. Proliferation of cancer cells is suppressed and cancer cell death is induced. (2) Jab1 knockout cells and individual animals were prepared and analyzed for various functions. It has been found that it is deeply involved in a wide range of life phenomena such as development, cell proliferation, cell death, and cell cancer, and (3) that the inflammatory response is promoted in cells in which Jab1 expression has been reduced by the knockout method. The present invention has been completed.

  That is, the present invention includes the following inventions A) to L) as industrially and medically useful inventions.

  A) A method of regulating the growth, death, differentiation, or canceration of animal cells (particularly mammalian cells) by controlling the expression of Jab1 gene or the activity of Jab1 protein. For example, a cell growth suppression method by suppressing the expression of the Jab1 gene can be mentioned. The expression / activity control includes both enhancing and suppressing expression / activity.

  B) A method of suppressing cancer cell growth or inducing cell death of cancer cells by suppressing Jab1 gene expression or Jab1 protein activity. In fact, this method has been shown to inhibit the growth of various human cancer cell lines.

  C) A method of suppressing the growth of ES cells or other primary cells or inducing cell death of primary cells by suppressing Jab1 gene expression or Jab1 protein activity. Primary cells are not limited to universal cells, but have a broad meaning including all types of stem cells, mouse embryonic fibroblasts (MEF), undifferentiated cells, and other multipotent cells. This method is useful for, for example, a technique for suppressing undesired cell growth when a tissue / organ is produced using ES cells in regenerative medicine.

  D) A method of promoting proliferation of primary cells and other target cells by enhancing Jab1 gene expression or Jab1 protein activity. This method is useful for techniques for increasing the number of hematopoietic stem cells collected during bone marrow transplantation or umbilical cord blood transplantation, techniques for proliferating liver cells and using them for drug efficacy evaluation, ex vivo stem cell proliferation methods, and the like. Examples of methods for enhancing Jab1 expression / activity include a method of introducing a Jab1 expression vector into a cell, a method of administering a drug that activates Jab1, and the like.

  E) Jab1 knockdown cell in which expression of Jab1 gene is temporarily or continuously suppressed. It is only necessary that the expression level is substantially reduced as compared to the wild type, and Jab1 expression may not be completely suppressed.

  F) A Jab1-specific RNAi expression vector introduced into a cell in order to specifically suppress the expression of the Jab1 gene. In particular, it is useful for suppressing the intracellular expression of the Jab1 gene continuously and stably.

  G) A Jab1-modified cell obtained by modifying the Jab1 gene sequence in the genome, wherein the activity of the Jab1 protein is modified. Examples thereof include cells modified by introducing a mutation such as a point mutation into a region important for Jab1 protein activity and expressing a Jab1 mutant protein that has lost its original activity. It may be a cell modified so as to express a Jab1 mutant protein having an activity higher than the original activity.

  H) A Jab1-modified non-human animal obtained by modifying the Jab1 gene sequence in the genome, wherein the activity of the Jab1 protein is modified. Similar to the above, animals modified to express Jab1 mutant protein that has lost its original activity, and the like can be mentioned.

  I) A Jab1 knockout cell obtained by modifying all or part of a Jab1 gene sequence in a genome, wherein one or both Jab1 genes on a homologous chromosome are disrupted.

  J) A Jab1 knockout non-human animal obtained by modifying all or part of the Jab1 gene sequence in the genome, wherein one or both Jab1 genes on the homologous chromosome are disrupted. As described later, it can be used as an inflammatory disease model animal. Although knockout mice in which both Jab1 genes were disrupted were lethal during fetal life, fetal utilization is considered.

  K) A screening method for an anticancer agent characterized by searching for a Jab1 inhibitor that inhibits the activity or expression of Jab1. Any of in vitro and in vivo screening systems may be used. The development of highly versatile anticancer agents effective for various cancer cells can be expected.

  L) A method for screening an anti-inflammatory agent using the cell or animal according to any one of G) to J) above. Screening of anti-inflammatory agents at the cultured cell level and individual level is possible.

  The present invention is a method of regulating the growth, death, differentiation, or canceration of animal cells by controlling the expression of Jab1 gene or the activity of Jab1 protein, focusing on the function of Jab1 / COP9 signalosome. In the conventional method of controlling cell proliferation and the like by adding a signal factor such as cytokine from the outside, the factor acting on the target cell may differ, and it was necessary to change the signal factor according to the target cell. In this case, since Jab1 / COP9 signalosome is present in almost all mammalian cells, it is not necessary to change the material or method according to the target cell.

  In addition, in the conventional method for controlling the activity of a specific functional molecule such as a cell cycle regulator, there is a risk that an undesirable effect occurs. In the case of the present invention, since a plurality of functional molecules downstream of the Jab1 / COP9 signalosome can be controlled in a coordinated manner, it is possible to effectively generate a cellular response.

  In fact, the present invention was found to have a growth inhibitory effect on various human-derived cancer cell lines, and was found to be applicable to a wide range of cells and tissues.

Hereinafter, specific embodiments of the present invention will be described in more detail.
[1] Cell growth control method of the present invention The present invention artificially controls the expression of the Jab1 gene or the activity of the Jab1 protein, so that the growth, death, differentiation, or canceration of animal cells (particularly mammalian cells) is achieved. It is a method of adjusting. As described above, expression / activity control includes both enhancing and suppressing expression / activity. For example, a method of suppressing the expression level of the Jab1 gene in the target cell / tissue, and conversely, a method of increasing the expression level of the Jab1 gene can be mentioned. Examples of the method for suppressing the expression level of the Jab1 gene include a method for modifying the Jab1 genome (for example, a knockout method described later) and a method for suppressing after transcription (for example, a knockdown method by an RNAi method described later). Other methods may selectively inhibit Jab1 gene transcription, or selectively inhibit Jab1 splicing, translation, or post-translational modification processes to specifically suppress Jab1 protein expression. It may be a method to do. The Jab1 selective inhibitor may be a substance having an action of specifically inhibiting the expression of Jab1 protein in addition to a substance having an action of inhibiting the activity of Jab1 protein. The present invention also includes a method of specifically suppressing the expression of Jab1 gene / protein by administering such a Jab1 selective inhibitor.

  As a method for specifically suppressing the expression level of the Jab1 gene, the RNAi method may be used. For example, a method of introducing artificially produced siRNA into a cell, a method of producing a Jab1-specific RNAi expression vector, and introducing this into a cell are exemplified. The Jab1-specific RNAi expression vector is (1) designed to express dsRNA having a hairpin structure of an appropriate length with a single RNA in the target cell, (2) sense strand and antisense strand respectively That are designed to be expressed and associated in the target cell. The Jab1-specific RNAi expression vector used in the examples described later is the type (2) above. The sequence of siRNA is not particularly limited, and may be arbitrarily designed according to a known method. By using a Jab1-specific RNAi expression vector, the Jab1 gene can be knocked down stably and stably.

  Other methods for specifically suppressing the expression level of the Jab1 gene include, for example, a method of administering an antisense oligonucleotide, a ribozyme, a low molecular compound or the like to a target cell / tissue.

  Examples of methods for enhancing Jab1 expression / activity include a method of introducing a Jab1 expression vector into a cell, a method of administering a drug that activates Jab1, and the like. The Jab1 expression vector is not particularly limited, and an appropriate vector and Jab1 DNA may be determined according to the target cell / tissue (for example, mouse Jab1 cDNA (accession number: AF068223) can be used).

  Of course, the present invention is not limited to the above exemplified methods, and various conventionally known methods can be applied to control the expression of the Jab1 gene and the activity of the Jab1 protein. Further, a method newly developed after the present invention may be used.

  As shown in the examples described later, the research results obtained this time revealed that the functions of Jab1 are deeply involved in a wide range of life phenomena such as early development, cell proliferation, cell death, cell differentiation, and cell canceration. It was. Therefore, the effect of controlling Jab1 expression / activity can also be applied to a wide range of cell proliferation, death, differentiation, and canceration.

  Many diseases are caused by abnormalities in the mechanisms that regulate cell growth, death, differentiation, and canceration. For example, cancer is a representative example. The present invention can be used for development of diagnostic methods and therapeutic methods for such diseases, screening for therapeutic agents, and the like.

  For example, this study and experiment revealed that reducing Jab1 expression suppresses the growth of various cancer cells and causes cell death. Therefore, the present invention is useful for the development of anticancer agents. Suppression of Jab1 expression was not limited to solid tumors such as pancreatic cancer, breast cancer, and cervical cancer, but was also effective against leukemia (lymphoma / hematologic malignancy) and teratoma (teratomas). Therefore, the development of anticancer agents effective against various types of cancer can be expected. In particular, since an antiproliferative effect on pancreatic cancer was observed, development of an effective anticancer agent against pancreatic cancer can be expected.

  The present invention can also be used for target (primary) cell amplification methods and the like. For example, it is conceivable that hepatocytes are proliferated by the method of the present invention and used for drug efficacy evaluation or for ex vivo stem cell amplification methods.

  Furthermore, as shown in the examples described later, since the inflammatory reaction is promoted in Jab1 +/− cells (mouse), the present invention is also applicable to the development of diagnostic methods and therapeutic methods for various inflammatory diseases, screening of therapeutic agents, and the like. Is available.

[2] Jab1 gene-modified cells and animals of the present invention For example, Jab1 knockout animals, ie, obtained by modifying all or part of the Jab1 gene sequence in the genome, wherein one or both Jab1 genes on the homologous chromosome are Disrupted non-human animals can be generated using gene targeting methods.

  The gene targeting method may be performed according to a normal method. For example, first, a targeting vector (targeting construct) for homologous recombination is constructed. The targeting vector can be prepared by a known method. In general, a Jab1 genomic DNA fragment, a commercially available plasmid, a marker for positive selection (eg, PGK-neo cassette), and a marker for negative selection (DTA gene, HSV- Each fragment such as a tk gene) can be appropriately ligated. At this time, the targeting vector may be designed so that the target restriction enzyme cleavage site is arranged at an appropriate position. Moreover, since the efficiency of targeting depends on the length of the homologous region, it is preferable that the homologous region is as long as possible. Furthermore, since the targeting vector is preferably linear rather than circular, an appropriate restriction enzyme cleavage site may be provided in one part other than the homologous region for linearization.

  The targeting vector prepared by the above method is introduced into totipotent cells derived from the target animal such as ES cells by electroporation and the like, and then the cells in which the desired homologous recombination has occurred are selected. The screening can be efficiently performed using a drug by a positive-negative selection method. After selection, the cells in which the desired homologous recombination has occurred are confirmed by Southern blotting or PCR. The totipotent cells finally confirmed to have homologous recombination are introduced into blastocysts (blast cysts) collected from the uterus during pregnancy. The introduction of cells into the blastocyst can be performed by a microinjection method or the like, but is not limited thereto.

  The blastocyst is transplanted to a temporary parent according to a conventional method. By crossing a germline chimeric animal (preferably male) born from a foster parent with a wild type animal (preferably female) having a wild type Jab1 gene homozygously, as a first generation (F1), A heterozygous individual (and cell) in which one Jab1 gene is disrupted by homologous recombination can be obtained. Furthermore, by mating these heterozygous individuals, a homozygote in which both Jab1 genes on the homologous chromosome are disrupted can be obtained as the second generation (F2) (although it is lethal in the fetal period in mice). Met).

  The gene targeting method described above is merely an example, and it goes without saying that various known modifications can be made.

  The target animals of the Jab1 knockout animals are not particularly limited, and examples thereof include mammals such as cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, and rats. Of these, rabbits, dogs, cats, guinea pigs, hamsters, mice, and rats are preferred for use as experimental animals, of which rodents are more preferred, and many inbred strains have been produced. Particularly preferred are mice that are equipped with techniques such as in vitro fertilization. As totipotent cells, in addition to fertilized eggs and early embryos, pluripotent ES cells and cultured cells such as somatic stem cells can be targeted.

  Other Jab1 gene-modified cells and animals can also be produced by various gene manipulation techniques similar to those described above or conventionally known.

  Of course, the Jab1 gene-modified cells and animals of the present invention, including Jab1 knockout animals, can be used for functional analysis of Jab1, but as described above, the functions of Jab1 are early development, cell proliferation, cell death, It is thought to be deeply involved in a wide range of life phenomena such as cell differentiation and cell cancer. There are many diseases caused by abnormalities in the mechanisms regulating cell proliferation, death, differentiation, and canceration. Therefore, mice and cells in which the Jab1 gene is disrupted / modified can be used for the development of diagnostic methods and therapeutic methods for such diseases and screening for therapeutic agents. In particular, heterozygous mice in which one of the Jab1 genes is disrupted have an accelerated inflammatory response, as shown in the examples below. Can be induced and can be an unprecedented inflammation model mouse.

[3] Screening method of the present invention When Jab1 was knocked down by RNAi method, cell death was induced in various human cancer cells. Therefore, a Jab1 inhibitor that suppresses or inhibits the activity or expression of Jab1 can be expected to be used as an anticancer agent (or a lead compound thereof) effective for various cancer cells, and a screening method thereof is also included in the present invention. It is.

  As the screening method of the present invention, various conventionally known methods for examining gene / protein expression levels, changes in protein activity, and the like can be applied and are not particularly limited. Moreover, you may use the screening method newly developed after this invention. Any of in vitro and in vivo screening systems may be used, and screening may be performed with a cell-free system. The Jab1 gene / protein may be derived from mice or other animals in addition to those derived from humans. Of course, screening may be performed using information on the higher-order structure of the Jab1 protein. Examples thereof include a screening method by detecting activated Jab1 by non-denaturing electrophoresis.

  The method for screening an anti-inflammatory agent using the Jab1 gene-modified cell or animal described in [2] above is also included in the present invention. Also in this screening, the method is not particularly limited, and screening of anti-inflammatory agents is possible at the cultured cell level and the individual level.

  Examples of the present invention will be described below with reference to the drawings, but the present invention is not limited to these examples.

[Example 1: Induction of cancer cell death by Jab1 knockdown]
Study whether the growth of cancer cells can be suppressed by preparing RNAi expression vectors specific for mouse and human Jab1, introducing this vector into various human cancer cell lines, and suppressing the expression level of Jab1 did.

The RNAi expression vector was prepared using pSilencer expression vector system (Ambion). The sequence inserted into the vector is for human Jab1,
hJab1-1 sense (5'-GCT CAG AGT ATC GAT GAA ACG AGT CTC ATA GCT ACT TTT TTT TT-3 ') (SEQ ID NO: 1)
hJab1-1 anti-sense (5'-AAT TAA AAA AGC TCA GAG TAT CGA TGA AAT CTC TTG AAC GAG TCT CAT AGC TAC TTT GGC C-3 ') (SEQ ID NO: 2)
Or
hJab1-2 sense (5'-ATA CGA CAA GAA ACA GCA GTT CAA GAG ATA TGC TGT TCT TTG TCG TCT TTT TT-3 ') (SEQ ID NO: 3)
hJab1-2 anti-sense (5'-AAT TAA AAA AAT ACG ACA AGA AAC AGC AGT CTC TTG AAT ATG CTG TTC TTT GTC GTC GGC C-3 ') (SEQ ID NO: 4)
And for mouse Jab1,
mJab1-1 sense (5'-CAA CAA CAA GAA ATC CTG GTT CAA GAG ACC AGG ATT TCT TGT TGT TGT TTT TT-3 ') (SEQ ID NO: 5)
mJab1-1 anti-sense (5'-AAT TAA AAA ACA ACA ACA AGA AAT CCT GGT CTC TTG AAC CAG GAT TTC TTG TTG TTG GGC C-3 ') (SEQ ID NO: 6)
It is.

The human cancer cell line introduced with the above vector is a cell line derived from the following solid tumor.
MIAPaCa: Human pancreatic cancer cell line
PANC1: Human pancreatic cancer cell line
MCF7: Human breast cancer cell line
HeLa: Human cervical cancer cell line
293: Human fetal kidney-derived cancerous cells

  Lipofectamine method was used for the intracellular introduction of Jab1-specific RNAi expression vector.

  The experimental results are shown in FIG. As shown in the figure, as a result of suppressing the expression of Jab1 with Jab1 RNAi for all the above human cancer cell lines, the growth of cancer cells was suppressed and cancer cell death was induced.

[Example 2: Disruption of Jab1 gene by targeting]
First, the genomic structure of mouse Jab1 was analyzed. The result is shown in the upper part of FIG. 2 (Wild-type locus). In the figure, black boxes indicate exon coding regions, white boxes indicate non-coding regions, and H, N, and V indicate restriction sites of HindIII, NheI, and EcoRV, respectively.

  The structure of the prepared targeting vector is shown in the middle of the figure. In the figure, NEO represents a neomycin phosphotransferase gene, and TK represents a thymidine kinase gene. The vector is constructed by first subcloning a genomic DNA fragment of about 1 kb upstream of the starting methionine and a genomic DNA fragment of about 5 kb downstream of exon 6 and then putting these fragments into the EcoRI and XhoI sites of the ploxPNT vector. Each inserted. Thereafter, the vector was linearized at the XhoI site and introduced into mouse RF8 ES cells by electroporation.

  ES clones that had undergone the desired homologous recombination by targeting were selected with 200 μg / ml G418 and 0.2 μM FIAU, and further confirmed by Southern blot analysis. Two types of probes, probe A and B, were used (see the upper part of FIG. 2). In the case of probe A, fragments were detected after digesting the genomic DNA with HindIII, and in the case of probe B, the fragments were detected after digesting the genomic DNA with NheI and EcoRV. When homologous recombination occurs, a 1.6 kb fragment is detected in probe A and a 6.6 kb fragment is detected in probe B, as shown in the lower part of the figure (Targeted locus). FIG. 3 shows the result. In the figure, “+ / +” indicates a sample having a wild-type Jab1 homozygote, and “+/−” indicates a sample in which one Jab1 of a homologous chromosome is destroyed by targeting (hereinafter the same).

  Jab1 +/− ES cells determined to have a genotype “+/−” by the above analysis were microinjected into C57BL / 6 mouse blastocysts. The resulting chimeric male mice were mated with C57BL / 6 female mice, and the genotype of the offspring was determined by genomic PCR analysis using Jab1-specific primers and NEO-specific primers. Each region of the primers a, b, and c used is shown in FIG. FIG. 4 shows the results of PCR analysis of genomic DNA isolated from cultured blastocysts. In the case of the wild type allele (WT), a 2047 bp band is detected, and in the case of the Jab1 disrupted allele (KO), a 2464 bp band is detected. In the figure, “− / −” indicates a sample in which both Jab1 of homologous chromosomes are destroyed by targeting (the same applies hereinafter).

[Example 3: Histological analysis of normal Jab1 fetus and Jab1 mutant fetus]
Homo-disrupted mice (-/-) that destroyed both Jab1 alleles were lethal during the fetal period (around 5 days of embryonic life).

  Each molecule was detected in Jab1 normal fetus and Jab1 mutant fetus by immunohistochemistry.

  A uterus was removed from a pregnant female mouse, fixed with 4% paraformaldehyde overnight, embedded in paraffin, a 4 μm-thick fetal section was prepared, and stained with hematoxylin and eosin. Subsequently, each molecule was detected using a plurality of antibodies by immunohistochemistry. In addition, TUNEL staining was performed on fetal slices to detect apoptosis. The result is shown in FIG.

  Rabbit polyclonal antibodies were used for Jab1, CSN1, cyclin E, and Cul1 antibodies (αJab1, αCSN1, αCycE, αCul1), and mouse monoclonal antibodies were used for p27 and p53 antibodies (αp27, αp53). In the figure, a and b are sections of E7.5, cr is a section of E6.5, ad is a result of hematoxylin and eosin staining, ep is a result of immunostaining with the above antibodies, and q and r are results of TUNEL analysis It is. “+” Indicates the result of a normal Jab1 fetus, and “− / −” indicates the result of a Jab1 homozygous fetus.

  In the Jab1 homo-disrupted fetus “− / −”, expression of CSN1, which is another component of Jab1 and COP9 signalosome (CSN), was decreased, while p27, p53 and cyclin E were expressed relatively strongly. Apoptosis was also observed.

  In a similar experiment, CSN3 and CSN8, which are other components of CSN, were examined, and in both cases, a decrease in expression was observed in the Jab1 homo-disrupted fetus “− / −”.

[Example 4: Analysis of cultured cells from blastocysts]
E3.5 blastocysts of Jab1 normal type (+ / +) and Jab1 mutant type (-/-) were isolated from uterus, cultured in ES medium in vitro, and each molecule was detected by immunofluorescence analysis. It was. The result is shown in FIG.

  In the figure, a and f are the results of observing cells cultured for 24 hours, b, g and c and h are the results of observing cultured cells for 72 hours and 120 hours, respectively, and d and i are uptakes of bromodeoxyuridine (BrdU). As a result of examining using an anti-BrdU antibody, k and r were cultured for 72 hours and observed with a phase contrast microscope. E and j were examined for apoptosis of the cultured cells by TUNEL assay. (L, s) , (m, t), (n, u), (o, v), (p, w), (q, x) are also Jab1, CSN1, cyclin E, p27, The results of detecting p53 and Cul1.

  Similar to the results of Example 3, in the Jab1 mutant (− / −), Jab1 and CSN1 expression was decreased, while p27, p53 and cyclin E were expressed relatively strongly. In addition, a decrease in proliferation ability was observed, and apoptosis was observed.

[Example 5: Study on growth of Jab1 +/− mice and cells]
Heterogeneous mice that destroyed one of the Jab1 alleles (+/−) grew to adults and had the ability to reproduce, but were smaller than the wild-type (+ / +) of the same litter (FIG. 7). The results of measuring the body weight of wild type (+ / +) and heterozygous mice (+/−) are shown in FIG.

  Next, primary mouse embryonic fibroblast (hereinafter referred to as “MEF”) is isolated from wild-type (+ / +) and heterozygous mouse (+/−) fetuses (embryonic day 14.5). The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), and the number of cells was counted over time. The result is shown in FIG. In the figure, the broken line is the result of Jab1 +/− MEF, and the proliferation ability was lower than that of the wild type (+ / +).

  FIG. 10 shows the results of immunoblot analysis performed on Jab1 + / + MEF and Jab1 +/− MEF. Each molecule was detected by Western blotting or non-denaturing electrophoresis. As a result, expression of p27 was strongly detected in Jab1 +/− MEF, while the amount of small complex containing Jab1 was decreased.

  FIG. 11 shows the proportion of cells that were cultured in Jab1 + / + MEF and Jab1 +/− MEF under the conditions of 0.1% FBS, synchronized with G0 / G1 phase, restimulated with 10% FBS, and then transferred to S phase. Is a result of examining the change by flow cytometry (in the figure, the broken line indicates the result of Jab1 +/− MEF). FIG. 12 shows the results of immunoblot analysis of p27 expression in each MEF after restimulation with 10% FBS. In Jab1 +/− MEF, p27 could not be sufficiently downregulated in the G1 phase, and the transition from the G0 phase to the S phase was delayed as compared with the wild type.

  Next, a teratoma formation experiment was conducted. Jab1 + / + ES cells and Jab1 +/− ES cells were injected subcutaneously into nude mice, and after 16 days, the formed teratomas (tumors) were excised and weighed. As a result, as shown in FIG. 13, in Jab1 +/− ES cells, the weight (and size) of the formed tumor was small, and a tumor suppressing effect was observed.

  FIG. 14 shows the results of immunoblot analysis performed on Jab1 + / + ES cells and Jab1 +/− ES cells. Each molecule was detected by Western blotting or non-denaturing electrophoresis. As a result, in Jab1 +/− ES cells, a significant decrease in the expression of a small complex containing Jab1 was observed.

[Example 6: Study on canceration of Jab1 +/− mouse bone marrow cells]
Hematopoietic cells were isolated from bone marrow of Jab1 +/− mice and wild type (Jab1 WT) of the same litter, and Bcr-Abl was introduced in vitro and cultured in vitro. Bcr-Abl is a causative factor of chronic myelogenous leukemia (CML), and it is known that when a gene is introduced, cells become cancerous and cause abnormal growth. As a result of examining the frequency of cell proliferation over time after the introduction of the Bcr-Abl gene, as shown in FIG. 15, it was found that canceration of cells was suppressed in Jab1 +/− mouse bone marrow cells.

[Example 7: Promotion of inflammatory reaction in Jab1 +/- cells]
When Jab1 +/− mouse bone marrow cells were cultured for a long period of time, and the resulting hematopoietic cells were observed under a microscope, generation of toxic granules (Toxic granule) was observed as shown in FIG. Jab1 +/− mouse ES cells were induced in vitro, and toxic granules were also observed in the resulting hematopoietic cells. From this result, it is considered that in Jab1 +/− cells (and mice), the decrease in Jab1 promotes the inflammatory response.

  As described above, the present invention relates to a method for regulating the growth, death, differentiation, or canceration of animal cells by controlling the expression of Jab1 gene or the activity of Jab1 protein. In addition to being used for various purposes for the purpose of inhibiting or promoting proliferation, it can be widely used in industries such as the development of anticancer agents and the development of anti-inflammatory agents.

It is a graph which shows the experimental result which investigated the proliferation inhibitory effect of the cancer cell by Jab1 knockdown. It is a figure explaining the gene targeting method of a present Example. It is a figure which shows a Southern blot analysis result. It is a figure which shows a genomic PCR analysis result. It is a figure which shows the histological analysis result of a Jab1 knockout mouse (fetus). It is a figure which shows the result of having culture | cultivated and analyzed the blastocyst of the Jab1 knockout mouse (fetus). It is a figure which shows the magnitude | size of a Jab1 +/- mouse | mouth compared with a wild type. It is a graph which shows the body weight measurement result of a Jab1 +/- mouse | mouth and a wild type. It is a graph which shows the result of isolating and culturing MEF from Jab1 +/− mice and wild-type fetuses, and counting the number of cells over time. It is a figure which shows the result of having performed the immunoblot analysis with respect to Jab1 +/- mouse and wild type MEF. It is a graph which shows the result of having investigated the time-dependent change of the cell ratio which stimulated Jab1 +/- and wild-type MEF synchronized by the low serum state by serum re-addition, and moved to S phase after that by the flow cytometry method. It is a figure which shows the result of carrying out the immunoblot analysis of the expression of p27 in each MEF after re-adding serum. It is a graph which shows the result of having conducted the teratoma formation experiment. It is a figure which shows the result of having performed the immunoblot analysis with respect to Jab1 +/- mouse | mouth and a wild type ES cell. It is a graph which shows the examination result regarding canceration of a Jab1 +/- mouse | mouth bone marrow cell. It is a figure which shows that the inflammatory reaction is accelerated | stimulated in Jab1 +/- cell compared with a wild type. It is a figure explaining the function and action of Jab1 / COP9 signalosome.

Claims (5)

A method for screening an anti-inflammatory agent using a Jab1 knockout cell obtained by modifying all or part of a Jab1 gene sequence in a genome and disrupting one or both Jab1 genes on a homologous chromosome. A method for screening an anti-inflammatory agent using a Jab1 knockout non-human animal obtained by modifying all or part of a Jab1 gene sequence in a genome and having one or both Jab1 genes on a homologous chromosome destroyed. A method of regulating proliferation or death of ES cells by controlling expression of Jab1 gene or activity of Jab1 protein. A method of suppressing the proliferation of ES cells by suppressing the expression of Jab1 gene or the activity of Jab1 protein. A method of promoting ES cell proliferation by enhancing Jab1 gene expression or Jab1 protein activity.