WO2006090679A1 - METHOD FOR DETECTION OF DNA DAMAGE USING Frag1 GENE OR GENE PRODUCT THEREOF AS MARKER - Google Patents

Abstract

Disclosed are a novel molecular marker which enables to detect a DNA damage or unscheduled replication of DNA which may directly cause a genetic change or the like, with high sensitivity, and the like. A method for detecting a DNA damage by using, as a marker, a Frag1 gene, a gene product of the gene or a phosphorylated product of the gene product or a cellular response associated with the gene or product in a sample; a method for screening for an exogenous stimulation involving any one of various carcinogenic substances including various DNA replication inhibitors, carcinogens and potential carcinogens or for an anti-cancerous agent; a method for testing or diagnosing a disease associated with the expression or activity of Frag1; a kit for use in these methods; and the like.

Description

 Specification

 DNA damage detection method using Fragl gene or its gene product as a marker

 Technical field

 [0001] The present invention relates to a method for detecting DNA damage (unscheduled-replication or inhibition of replication) using the Fragl gene, which is a care taker gene involved in tumor suppression, and its gene product as a marker. In particular, the present invention relates to a highly sensitive detection method for DNA damage in the S phase of the cell cycle.

 Background art

 [0002] Accumulation of genomic damage regions associated with chromosomal location abnormalities during tumor growth process activates proto-oncogenes and inactivates tumor suppressor genes (non-patent literature)

Cancer cells can be analyzed by genome-wide analytical techniques such as Doo chromosome painting, chromosome comparison annoyance, representational differential analysis, restriction landmark genome scanning, and high-throughput analysis of lack of heterozygosity. As a result of the instability of the genome, it became clear that a great deal of chromosome damage accumulated. Therefore, in order to identify the affected genes and elucidate their role in tumorigenesis, it is necessary to distinguish important genomic damage from other noises.

[0003] It has been proposed to classify tumor suppressor genes into two types, "gate keeper" and "care taker" (Non-patent Document 2). In contrast to gatekeepers, caretaker genes do not directly control cell growth and function to prevent or prevent genomic destabilization. This function works directly or indirectly in the repair of DNA strand breaks by the homologous recombination pathway. Such genomic instability can be attributed to carcinogenic susceptibility clinics such as telangiectatic movement disorders, hereditary breast cancer, Bloom's syndrome, and Werner's syndrome. Related to genetic syndrome. Accumulation of genome-wide caretaker abnormalities, in cooperation with the mutated gatekeeper, can cause severe lesions, resulting in transformed cells that have acquired selective growth and viability. [0004] Large scale chromosomal rearrangements can result from improper repair of DNA double-strand breaks (DSBs). These DSBs are the primary cause of chromosomal abnormalities and can occur when disrupted by the natural order of DNA replication. DSBs can lead to cell death, or if transection, inversion, amplification, and deletion in daughter cells are possible if they are viable by cleavage or do not immediately meet cell death It leads to chromosomal abnormalities. Although replication stress can cause replication forks to slow down or stop, natural sequences such as replication fotenoria and replication slow zones known in yeast research can also cause such a stop. These events trigger the activation of cell checkpoints, giving them time to repair the damage before the cell cycle progresses. Therefore, elucidating processes in slowed or stopped replication forks is important to understand replication-faithful regulatory mechanisms to prevent carcinogenesis involving DNA replication damage. However, the mechanism by which DNA damage induces apoptosis is not yet fully understood (Non-patent Document 3).

 [0005] Disrupted stress on individual cells due to the extracellular environment or intracellular physiological activity prevents cell cycle progression, and the cellular response to it regulates DNA repair, and Apoptotic bow through transmembrane cell death-inducing receptors and mitochondrial cell death induction pathways I activate checkpoints that activate the signal pathway DNA out of the damage stress caused by this extracellular or intracellular event The mitochondrial cell death induction pathway plays an important role in the induction of cell death as one of the cellular responses to genotoxic stress (replication stress) that induces damage. Bcl2 family proteins are the most important functional molecules that play an important role in this mitochondrial cell death induction pathway. This Bcl2 family protein has two groups of anti-apoptotic proteins and apoptosis-inducing proteins that have two opposing functions. Examples of anti-apoptotic proteins are Bcl2 and Bel-X. Examples of apoptosis-inducing proteins are Bax and Ba

d. The results of the functional balance of these Bcl2 family proteins are thought to play a fate-determining mechanism for cell death induction as a whole cell. Among the cell death induction mechanisms for genotoxic stress that induces DNA damage, DNA damage force to mitochondria The matching part is left with many unknown points (Non-Patent Document 4).

 Rad9, a protein involved in the regulation of DNA damage induction checkpoints, has been identified in the present invention as a downstream molecule of the Fragl function. This Rad9 is a force that interacts with this anti-apoptotic Bcl2 family protein Bcl2 and Bcl-X

 Shi

It does not react with the Bax and Bad protein-promoting proteins. Studies in yeast and humans have shown that Rad9, Husl and Radl function as PCNA-like sliding clamps as heterotrimeric complexes, or “9-1-1 complexes”. In response to DNA damage, the 9-1-1 complex binds around DNA lesions via Radl 7, which promotes phosphorylation and activation of Chkl kinases via Atr. Human Radl7 protein was shown to bind to chromatin before injury and to capture Rad9 protein on chromatin after injury. Combined with recent reports that the 9– 1– 1 complex is also involved in DNA repair, the 9–1 1 complex coordinates multiple functions of checkpoint activation, DNA repair and apoptosis. It is suggested that!

Non-patent literature l: Hanahan, D., Weinberg, R.A.The hallmarks of cancer, Cell 100, 1, 5 7-70, 2000.

Non-Patent Document 2: Levitt, NC, Hickson, ID, Trends Mol Med., 2002. 8 (4): p.179-86 Patent Document 3: Russell, P., checkpoints on the road to mitosis.Trends Biochem bci. , 1998. 23 ((10)): p. 399-402.

 Patent Document 4: Adams, J.M., Cory, S. The Bel— 2 protein family: arbiters of cell survi val. Science281 (5381) 1322-6, 1998

Disclosure of the invention

Problems to be solved by the invention

As already mentioned, checkpoints are made when genomic changes occur temporarily due to exogenous / intrinsic replication stress at an earlier stage of chromosomal / genetic change (ie, very early oncogenesis). Activation, slowing down and halting the cell cycle repairs genomic damage, but dangerous mutant cells are removed by apoptosis if they result in incomplete repair or repair failure. The present inventor newly elucidated that the Fragl gene plays a central role in this cell fate decision mechanism, and changed the expression of the Fragl gene and the gene product. It was found that DNA damage that directly causes gene changes can be detected with high sensitivity by detecting changes in protein phosphate and protein phosphate.

[0007] That is, as specifically shown in the following examples, the present inventor has found that when DNA damage such as replication inhibition occurs when some exogenous or endogenous replication stress is applied to cells and replication damage occurs. 1 is regulated and Rad9-Frag 1 binding is dissociated via Atr-induced Frag 1 phosphate (2 sites) and dissociated Rad9 binds to anti-apoptotic Bcl2 and apoptosis via Bax It was clarified to induce (see Fig. 11). The present invention has been completed based on such knowledge.

[0008] Accordingly, one object of the present invention is to provide a new molecule that enables highly sensitive detection of DNA damage or unscheduled-replication of DNA that directly causes genetic changes. Is to provide a marker. Yet another objective is to use this molecular marker to detect the "karakuri" where damaged cells survive non-physiologically at the divide of carcinogenesis at the individual level, medical research, diagnosis of precancerous lesions, carcinogenesis of substances. For the purpose of sex assessment (including potential non-genomic toxic substances in addition to genomic toxic substances), setting carcinogen thresholds, and detecting environmental carcinogens with similar action points with high sensitivity. Is to provide various methods.

 Means for solving the problem

That is, the present invention includes at least the following aspects.

 (1) A method for detecting DNA damage, which uses as a marker the Fmgl gene in the subject, the gene product or a phosphate thereof, or a cellular response involving them.

 (2) Screening for exogenous carcinogenic stimuli containing various carcinogenic substances such as various DNA replication inhibitors, carcinogens and potential carcinogens, or anticancer drugs using the method for detecting DNA damage of the present invention Method.

 (3) A method for examining or diagnosing a disease, cancer, or cancer cell involving Frag 1 expression or activity, using the method for detecting DNA damage of the present invention.

(4) DNA damage detection method, screening method, test or diagnosis method of the present invention Kit for use in.

 (5) An antibody that specifically recognizes the Fragl gene product, its phosphate, or a partial polypeptide thereof.

 (6) Nucleic acid molecules that hybridize under stringent conditions with the Fragl gene or part thereof, or their complementary strands.

 (7) A knockout or knockdown animal obtained by genetic engineering manipulation that lacks Fmgl gene or inhibits or suppresses Fragl gene expression and can show a high cellular response to DNA damage (human Excluded) or cultured cell lines established therefrom.

(8) Transgenic animals (excluding humans) or cultured cell lines established therefrom, in which a gene encoding a mutant of Fragl or a part thereof is introduced to change the detailed response to DNA damage.

 The invention's effect

 [0010] By using as a marker the Fragl gene, the gene product, or a phosphoric acid derivative of the gene product, or the cellular response in which they are involved in a subject as a marker, the cause of a direct change in detection of DNA damage DNA damage or unscheduled replication of DNA can be detected with high sensitivity. In particular, by using various transformed cells (transformants) or genetically modified animal systems such as knockout or knockdown animals of the present invention, the S phase of the cell cycle (especially the middle phase of the S phase, In the mid-S phase, DNA damage can be detected with high sensitivity. Further provided are antibodies and nucleic acid molecules for use in the detection, testing or diagnostic methods and kits of the invention.

 [0011] Further, by utilizing the method and kit of the present invention, it is possible to detect a “karakuri” in which damaged cells survive non-physiologically at an individual level in a watershed of carcinogenesis, medical research, precancerous lesions Diagnosis, evaluation of carcinogenicity of substances (in addition to substances with genomic toxicity (replication stress factor), including potential substances without genomic toxicity), setting of carcinogen thresholds, environmental carcinogens with similar action points Can be detected with high sensitivity.

 Brief Description of Drawings

[0012] FIG. 1 shows the results of an expression analysis of the Fragl gene. Lane 1 is 10% FCS / DMEM medium, and Lane 2 is a negative control for cells cultured in 10% FCS / DMEM medium. Added as an experiment). (A) RNA blot analysis. Cells synchronized in cell cycle with double thymidine block were treated with affedecolin (0.4 M), 20 g of total RNAs were separated in a gel, transferred to a membrane, and hybridized with a probe. Two probes, Fragl / N and Fragl / C, are also shown in C. (B) Expression of the Fragl gene in various cells. Lane 1, PU5 -1.8 (lymphoid tumor); Lane 2, RAW264.7 (leukemia virus-induced tumor); Lane 3, K-BALB (Kirsten murine leukemia virus-transformed fibroblasts; Lane 4, 3T3 (fibroblasts) ); Lane 5, LM (mouse L cells, transformed adipose tissue cells); Lane 6, P19 (teratomas); Lane 7, Hepa 1-6 (liver cancer); Lane 8, Rl.l (T cell lymphoma) Lane 9, L1210 (lymphoid leukemia); Lane 10, P388D1 (lymphoma); Lane 11, P815 (plasmacytoma); Lane 12, NB41A3 (neuroblastoma) (C) Fragl fragment: Fl, F2, Illustration of F3, F4, FZ Rb binding motif, LxCxE, two Atr phosphorylation sites are shown The positions of the two cDNA fragments obtained by clawing are counted from the first methionine. -1835 (= Fragl / N) and 3750-53 88 (= Fragl / C), and the position of the cDNA fragment used in the hybridization experiment is 1580-1830 And 5130-5380 NLS: Nuclear localization signal motif.

FIG. 2 shows the biological effect of down-regulation of Fragl gene expression due to replication stress. (A) Down-regulation of Fragl gene expression by Fragl siRNA. Mouse fibroblasts were transfected with a Fragl siRNA U6 expression vector and cultured in puromycin lug / ml selective medium. RNAs and proteins were extracted and analyzed by RT-PCR and Western blot. The experimental results of two independent clones are shown (# 403 and # 406). C1 and C2 are from cells into which control siRNA has been introduced. (B) Colony survival test after MMS exposure. Cell cycle synchronized to mid S phase (right) or not synchronized (left) lxlO ⁶ cells were subjected to double thymidine block, then cultured for 2 hours in thymidine-free medium, and cell cycle was put into S phase . After incubation for 1 hour with the indicated concentration of MMS, the MMS was washed away. Radiation was delivered at the indicated dose (bottom). As a colony survival test, cells were embedded in 1.5% methylcellulose and cultured for 10 days to observe the formed colonies. Viability for untreated cells was shown. a and b show the results of two independent experiments (參). w indicates wild type control cells (◯). [3] Fragl plays a role in the response to genomic toxins (replication stress). (A) Genome down-regulation of Fragl due to toxic stress. Mouse fibroblasts were treated with 0.4 nM affidocholine or 0.01% MMS for the indicated times. Cells were collected, proteins were extracted, and SDS-PAGE and Western blot were performed. (B) Down-regulation of Fragl enhances sensitivity to genomic venom stress. Two independent Fragl siRNA-introduced mouse fibroblasts (# 403 and # 406) were synchronized with the G1 cell cycle, cultured in thymidine-free medium, and MMS was observed. Cells were collected and proteins were extracted and subjected to SDS-PAGE and Western blot. Mismatch siRNA was used as a control (wild type control cells in FIG. 2).

[Fig. 4] Fragl is involved in the Rad9-Bcl2 pathway. (A) Co-immune sedimentation of Fragl, Rad9 and Bcl2. Mouse fibroblasts were treated with 0.4 nM alpha-choline or 0.01% MMS for 24 hours. Or it was exposed to ultraviolet rays (8 J / m ² ) and cultured for 24 hours. No processing on the left. Cells were collected and proteins were extracted and subjected to immunoprecipitation (IP) with anti-Rad9 antibody or normal rabbit serum (NRS), followed by Western blotting with anti-Fragle antibody, anti-Rad9 antibody, and anti-Bcl2 antibody. . (B) In vitro transcription / translation (IVTT) fragmentation of Fragl Fl, F2, F3, F4 and FZ protein fragments and GST_Rad9 binding protein. In vitro transcription translation (IVTT) was labeled with [ ³ ¾] methionine and incubated with GST-Rad9 binding protein. The conjugate was recovered by binding with glutathione agarose beads, subjected to SDS-PAGE, and exposed to film. The top is a withdrawal experiment. The lower left is Fragl Fl, F2, F3, F4 and FZ protein fragments that have been subjected to in vitro transcription translation (IVTT). Bottom right is Western blot with anti-GST antibody used in the experiment. (C) Colony survival assay of mouse fibroblasts after MMS exposure. Mouse fibroblasts were transferred to a cDNApcDNA expression vector incorporating Fl, F2, F3, F4 or FZ and cultured in G418 selective medium to perform a colony survival assay. (D) Cell death after MMS exposure. Hygromycin-resistant cells were prepared by introducing a Rad9 or Rad9-N terminal delta or Bcl2 plasmid together with a selection plasmid into the F2 gene-transferred cells. Viable cells were evaluated by Ellis Mouthsin B staining 24 hours after MMS exposure. Below are Western blots with anti-V5 tag (F2) antibody, anti-HA tag (Rad9) antibody, anti-HA tag (Rad9Ndelta) antibody and anti-Bcl2 antibody as shown. [Figure 5] Shows the role of Fragl in DNA damage response. (A) Mouse fibroblasts synchronized in cell cycle were cultured for the indicated time with or without MMS (0.01%). Western plots were performed with the indicated antibodies before and after immunoprecipitation with anti-Rad 9 antibody. (B) Synchronize the cell cycle of LxGxK, LxGxE or wild-type Fragl gene transducer with G1 and Western blot with anti-V5 (tag) antibody or anti-actin antibody before and after immunoprecipitation with anti-Rb antibody. Carried out. (C) One day after treating mouse fibroblasts with Atr siRNA, the cells were cultured for 4 hours with or without MMS (0.01%). Cells were collected and protein was extracted for immunoprecipitation or Western plotting. (D) Intracellular localization of Fragl and Rad9 after MMS treatment. After the MMS treatment, the cellular components were fractionated and subjected to Western blotting. P1, whole cell pellet; S2, cytoplasm and nucleus; P2, detergent-intolerant nucleus; S3, DNase I-extracted nucleus; P3, DNase I-resistant nucleus; S4, fraction containing chromatin; P 4, Nuclear matrix. CB, Coomassie brilliant blue staining (CBB); Western blots with anti-Grb2 and anti-Orc2 antibodies were performed as membrane or chromatin fraction controls, respectively.

 [Figure 6] Shows Fragl response to DNA damage. (A) Fragl and Rad9 association in DNA damage response. The cell cycle of the wild-type or mutant Fragl gene-transfectant was synchronized with G1, and MMS (0.01%) was added to the medium without addition of thymidine and cultured for 4 or 8 hours. Cells were collected, proteins were extracted, and Western plotting was performed with anti-V5 (tag) antibody or anti-actin antibody before and after immunoprecipitation with anti-Rb antibody. (B) Cell death of wild-type or mutant Fra gl transgenics after MMS exposure. Wild-type or mutant Fragl gene-transfectants were cultured for the indicated time with the addition of MMS (0.01%), and cell death was assessed by Ellis Mouthsin B staining.

FIG. 7 shows changes in expression of Fragl in response to DNA damage. The Fragl gene was identified, and the mRNA stability of the Fragl gene and the gene used for comparison was analyzed in the presence of aphidicolin.

 [Figure 8] Shows response to DNA damage and Fragl. (A) Expression change of Caspase7. (B) Change in expression of Bax and p53 (Serl5). (Right) Two independent MEFs forces The generated Fragl siRNA gene transfectants (# 403 and # 406) synchronize the cell cycle with G phase with double thymidine block, and release phosphate.

 1

After thymidine was washed in the impulse, put the cells at the indicated time. It was collected. Cell power proteins were extracted and Western blotted with anti-caspase7 antibody, anti-Bax antibody, and anti-p53 (Serl 5) antibody. (Left) Control. (C) p53-deficient mouse fibroblasts. The effect of Fragl downregulation on the release of cytochrome C from mitochondria was observed with a confocal microscope. Cells in which FRAG1 siRNA was introduced into mouse fibroblasts or control cells were cultured in the presence of 0.01% MMS for 2 hours. (Top and middle) After 24 hours, the cells were fixed, stained with anti-mitochondrial antibody (Mit.) Or anti-cytochrome C antibody (cyto.), And stained with secondary antibody. The white shadow is stained with antibodies. (Bottom) Overlapping two images.

 FIG. 9 shows Fragl, binding protein and cell death induction. (A) The immunoprecipitation method (IP) followed by the stamp lot method (wt). After 500 micrograms of the protein extract was shown and the antibody was immunoprecipitated (IP), the antibody was shown and the Western plot (Wt) was performed. (B) Confocal microscope. (Top and middle) Localization of Bax (top) and cytochrome c (middle) by immunofluorescence microscopy. F2 gene-introduced cells and controls were examined 24 hours before and after being placed in MMS-supplemented medium. (Bottom) Examination of Bax and cytochrome c. White V, shadow stained with antibody, where the presence of Bax and cytochrome c is localized.

[FIG. 10] The localization of Fragl and Atr after MMS exposure using a confocal microscope was observed over time. M EFs were cultured on a chamber bar slip and incubated in a medium containing 0.01% MMS for the indicated time. Subsequently, the cells were fixed with methanol and 0.05% Triton®-100, stained with the indicated antibody, and observed with a confocal microscope. (Upper and middle) Atr (upper) and Fragl (middle) focus formation were observed. (Bottom) An image of the top and the middle. A white shade shows the area stained with antibodies.

[Fig. 1l] A schematic diagram showing the Fragl function schema. Atr plays a direct or indirect role in Rad9 activity after replication inhibition. Fragl provides a place for Rad9 activity. Atr plays a role in the release of Rad9 from Fragl, so that Rad9 binds to the anti-apoptotic molecule Bcl2 to induce Bax-mediated apoptosis. Rb in the G1 phase of the cell cycle suppresses the progression of genomic abnormalities, exerts a negative control of the cell cycle, and as a result promotes cell survival. There, Rb binds to Fragl via the LxCxE motif on the Fragl protein and inhibits Fragl from activating Rad9. Rb progresses to S phase It is thought to be involved in the recruitment of Fragl to the site of DNA damage, which involves the activation of cytalin-dependent phosphorylase and the transcription factor E2fl.

 FIG. 12 shows the results of Western blotting using extracted anti-human Fragl antibody and anti-phosphorylated Fragl antibody.

 FIG. 13 shows an example of a vector used for production of a Fragl-deficient mouse.

 FIG. 14 shows an example of a vector used for production of a Fragl transgenic mouse.

 FIG. 15 shows a correlation between the tumor incidence in mice and the expression change (enhancement) of phosphate fragrance.

 FIG. 16 summarizes the correlation between the incidence of tumor tumors in mice, changes in the expression of Phosphate Fragl, and changes in the expression of Fhit gene and p53 gene.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0013] In the present specification, "Fragl gene, the gene product or a phosphate thereof, or a cellular response involving them" refers to the Fragl gene, the gene product, or the phosphate of the gene product. In a series of molecular interaction pathways involving rodents, molecular and cellular changes that occur around or downstream of their expression 'production etc., such as apoptosis, caspase protein and Bax It means the reaction and change in the cell such as activation of glycine, dissociation of Fragl and Rad9, and binding of Rad9 and Bcl2.

 [0014] In this specification, “DNA damage” refers to direct damage to DNA, such as DNA strand breaks, and the slowing or stopping of replication forks due to unscheduled-replication or polymerase inhibition. Inclusive of such replication inhibition, it generally means a state in which normal replication is inhibited depending on the degree to which DNA repair or apoptosis is directed. There are various types of stimulating DNA damaging stimuli, such as various carcinogenic substances such as various DNA replication inhibitors, carcinogens and potential carcinogens, as well as radiation, ultraviolet rays, etc. Other extrinsic carcinogenic stimuli such as electromagnetic waves and heat, or endogenous factors (such as replication stress) cause DNA damage.

[0015] As shown in the Examples below, the Fmgl gene has been identified by the present inventors from mouse cells under a replication inhibitory load and human cells by database search. Examples of the specific nucleotide sequence and the amino acid sequence encoded thereby include SEQ ID NO: 2 and SEQ ID NO: 3 (mouse Fragl gene), and SEQ ID NO: 5 and SEQ ID NO: 6 (human Fragl gene). Child). However, the biological origin of the gene is not particularly limited, and the gene is not limited to genes derived from mice and humans.

 [0016] The "Fragl gene product (protein)" in the present specification is substituted, deleted or added with one or several amino acids in the amino acid sequence specifically shown in the above SEQ ID NO. A protein (pyripeptide) having at least 90%, preferably 95%, more preferably 99% or more homology with the amino acid sequence and substantially the same activity as the Fragl protein included.

 [0017] Furthermore, the "Fragl gene" in the present specification is hybridized under stringent conditions with DNA containing a base sequence complementary to the base sequence specifically shown in the above SEQ ID NO. DNA (nucleic acid molecule) that encodes a protein (pyripeptide) having substantially the same activity as that described above is also included.

 [0018] In the present specification, "stringent conditions in DNA hybridization" are defined by an appropriate combination of salt concentration, organic solvent (for example, formamide), temperature, and other known conditions. . That is, stringency increases with decreasing salt concentration, increasing organic solvent concentration, or increasing hybridization temperature. In addition, the washing conditions after hybridization also affect stringency. This wash condition is also defined by salt concentration and temperature.

The stringency of washing increases with decreasing salt concentration and increasing temperature.

Accordingly, “stringent conditions” means the degree of homology between base sequences, for example, 90% or more, preferably 95% or more, more preferably 99% or more on the average on the whole. In addition, it means a condition in which a hybrid is specifically formed only between base sequences having high homology. Specifically, for example, the conditions include a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, and pH of 6 to 8 at a temperature of 60 ° C. to 68 ° C. A specific example of a stringent condition is a hybrid under conditions of 5 X SSC (750 mM NaCl, 75 mM trisodium citrate), 1% SDS, 5 X Denhardt solution 50% formaldehyde, and 42 ° C. A dialysis is performed, and washing is performed under the conditions of 0.1 X SSC (15 mM NaCl, 1.5 mM trisodium citrate), 0.1% SDS, and 55 ° C.

[0020] Hybridization is, for example, current 'protocorrez' in 'molecular ^ This method can be carried out according to a method known in the art, or a method analogous thereto, such as the method described in Current Protocols in Molecular Biology (edited by Frederick M. Ausubel et al, 1987). When using this library, it can be carried out according to the method described in the attached instruction manual.

As used herein, “homology” (or “identity”) refers to a chain between two strands in a polypeptide sequence (or amino acid sequence) or polynucleotide sequence (or base sequence). And the amount (number) of things that can be determined to be the same based on the matching relationship between each amino acid residue or each base, and the two polypeptide sequences or two It means the degree of sequence correlation between polynucleotide sequences. Homology can be easily calculated. Numerous methods for measuring homology between two polynucleotide or polypeptide sequences are known, and the term “homology” is well known to those skilled in the art. (For example, Les, AM (Ed .), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, DW (Ed.), Biocomputing: Informatics and Geno me Projects, Academic Press, New York, (1993); Grifin, AM & Grifin , HG (Ed.), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York, (19 87); Gribskov , M. & Devereux, J. (Ed.), Sequence Analysis Primer, M—Stockton Press, New York, (1991)). General methods used to determine the homology of two sequences are described in Martin, J. Bishop (Ed.), Guide to Huge Computers, Academic Press, San Di ego, (1994); Carillo, H & Lipman, D "SIAM J. Applied Math., 48: 1073 (1988), etc., but not limited to these. Preferred methods for measuring homology Such as those designed to obtain the largest fit between the two sequences to be tested, such as those assembled as a computer program. Preferred computer programming methods for measuring homology include the GCG program package (De vereux, J. et al "Nucleic Acids Research, 12 (1): 387 (1984)), BLASTP ゝ BLASTN ゝ F ASTA (Atschul, SF et al "J. Molec. Biol, 215: 403 (1990), etc. A method known in the art can be used without being limited to the above. [0022] In the present specification, "substantially equivalent activity" to Fmgl protein means that various activities such as physiological activity, biological activity and physical / physical activity of the protein are substantially equivalent or the same. It means that. Specific examples of such activity include, for example, the ability to bind to Rd9 and Rb, phosphorylation by Atr, and other functions in the apoptosis-inducing pathway as shown in the examples of the present specification. I can do it.

 [0023] The Fmgl gene (DNA) can be easily prepared by those skilled in the art based on the description in the present specification and known techniques in the technical field. For example, cloning using RT-PCR as described in the Examples, other ICACN (Isothermal and chimeric primer— initiated amplification of nucleic acids method, NASBA (Nucleic acid sequence based amplification) method, It can be easily prepared as cDNA by using any DNA amplification technology known to those skilled in the art such as TMA (Transcription-mediated amplification) and SDA (Strand Displacement Amplification) methods. These primers can be appropriately designed and selected based on the nucleotide sequence information of the Fmgl gene disclosed in this specification.

 [0024] In addition, the above DNA is obtained by a known method (for example, Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47: 411—418; Adams (1983) J. Am. Chem. Soc. 105: 661 ; Belousov (19 97) Nucleic Acid Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373—380; Blommers (1994) Biochemistry 33: 7886—7896; Narang (1979) Meth. Enzymol. 68: 90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; US Pat. No. 4,458,066). It can also be synthesized in vitro. It can also be produced by a method such as cleaving the polynucleotide of the present invention with an appropriate restriction enzyme. Furthermore, based on site-directed mutagenesis known to those skilled in the art, base mutations can be introduced into the DNA using a commercially available mutation system or the like.

[0025] Furthermore, the DNA encoding the Fmgl gene product (protein) thus obtained is converted into an appropriate recombination DNA such as a plasmid vector, a phage vector, and various hybrid vectors according to any method known to those skilled in the art. It can be inserted and incorporated into an appropriate expression vector, and an appropriate host cell can be transformed with the expression vector. [0026] This recombination DNA is any vector that can be handled by ordinary recombinant DNA techniques known to those skilled in the art. These vectors can be appropriately selected depending on the host cell to be introduced. The vector is introduced into a host cell to express the protein of the present invention transiently, or the whole or a part of the vector in one or more places in the genome of the host cell. Can be incorporated. As such a vector, various commercially available vectors known to those skilled in the art can be used.

 [0027] The expression vector of the present invention typically contains various promoters such as a constitutive expression promoter or various inducible expression promoters, various regulators such as enhancers and silencers, which are known to those skilled in the art. Various elements such as sequences, ribosome binding sites, signal sequences, translation initiation sequences, and other genes encoding foreign or endogenous proteins, various drug resistance genes, genes that complement auxotrophy, etc. This comes out.

 [0028] As host cells, prokaryotic microorganisms, eukaryotic microorganisms, plant cells, insect cells, avian cells, mammalian cells including primates such as humans and rodents such as mice, and the like can be used. For example, examples of prokaryotic microorganisms include Escherichia genus, Bacillus genus, or Streptomyces genus such as Streptomyces griseus or Streptococcus sericolor. Examples of eukaryotes include yeasts such as Saccharomyces and Pichia, Aspergillus genus such as Aspergillus oryzae and Aspergillus sau, Persyllium genus, Rhizopus genus, Metalithium genus, Monascus genus, Acremonium genus, and Mucor genus. The ability to select filamentous fungi such as, and basidiomycetes such as Trichoderma can also be selected. Examples of insect cells that can be used include Drosophila melanogaster and silkworm.

 [0029] The expression vector (DNA for recombination) containing the DNA of the present invention includes, for example, calcium chloride method, protoplast-PEG method, electopore position method, Ti plasmid method, particle gun method, baculovirus method, etc. It can be introduced into a host cell by any known method, and a transformant can be produced. Furthermore, the co-transformation method using multiple types of recombinant DNA is also possible.

[0030] Instead of the expression vector, the transformant of the present invention may be obtained using an appropriate DNA fragment itself containing a gene encoding the protein obtained by PCR amplification or the like. Is possible. In such a case, the composition can be used for transformation as a composition such as a solution optionally containing an appropriate buffer and other auxiliary agents in addition to the DNA fragment to be obtained.

 [0031] The transformant thus obtained is preferred for the production of the protein! / ヽ The protein is expressed by culturing under culture conditions under appropriate conditions, and by recovering the host cell and Z or medium strength. Gene products (proteins) can be produced. The medium used for culturing host cells should be appropriately selected from any medium known to those skilled in the art according to the configuration of the expression vector used (type of promoter, etc.) and the type of host. Can do.

 [0032] The above protein can also be produced using any in vitro transcription / translation system known to those skilled in the art using a rabbit reticulocyte extract or a wheat germ cell extract or the like.

 [0033] The thus produced Fragl gene product (protein) is an appropriate combination of any means known to those skilled in the art, for example, separation of medium and cells by centrifugation or filtration, and ammonium sulfate. Precipitation of the protein components of the medium with a salt-like salt, followed by hydrophobic chromatography, ion exchange chromatography, and affinity chromatography

Or by other chromatographic use. Alternatively, the protein of the present invention can be produced by a chemical synthesis method.

[0034] In the detection method of the present invention, DNA damage can be measured using any method known to those skilled in the art. For example, it can be detected by measuring the expression level of the Fragl gene as the amount of mRAN or cDNA, or by measuring the production amount of the Fragl gene product or its phosphate (protein or polypeptide). it can. It should be noted that these measurements do not necessarily need to be quantitative, and the effects of the present invention can be obtained sufficiently even with qualitative or semi-quantitative measurements by visual observation, depending on the specific measurement method and means. Can

[0035] The production amount of the Fragl gene product or its phosphate compound can be determined by, for example, immunostaining using an anti-Fragl antibody that specifically reacts with the protein, immunoprecipitation (pull-down assay), immunoblotting analysis, Western Use plot analysis and various immunological specific reactions such as EIA Detection method, amino acid sequence analysis method of gas phase sequencer etc. peptide using Edman method, and mass spectrometry represented by MALDI-TOFZMS and ESI Q-TOFZMS methods.

 [0036] The "anti-Flagl antibody" in the present specification can detect the Fragl gene product, its phosphate, or a partial polypeptide thereof as specifically shown in the following examples. Antibody. Therefore, in addition to antibodies specifically recognizing them, when they are bound to various labeling substances, antibodies that recognize such labeling substances are also included. Such an antibody can be obtained by using, for example, a protein prepared by the method as described above or an appropriate part thereof (partial polypeptide fragment) or various derivatives or complexes thereof as an antigenic substance or an immunogen. It can be prepared by an appropriate method known to those skilled in the art. For example, in the case of a polyclonal antibody, it can be administered to an appropriate animal such as mouse, rat, guinea pig, rabbit, goat, and chicken, and the antiserum can be prepared. Or, it is described in the monoclonal antibody production method (“monoclonal antibody”, Nagamune Kamei, Hiroshi Terada, Yodogawa Shoten, 1990; onoclonal Antioody James W. uoding, third edition, Academic Pres s, 1996) It can also be prepared as a monoclonal antibody by a known method using cell fusion. It should be noted that other antibodies used in the examples in the present specification can be easily prepared by those skilled in the art by the same method.

 [0037] It should be noted that the above-described antibody of the present invention can be obtained by genetic engineering (DNA recombination technology), for example, complete Fab, F (ab '), Fv fragment, etc., as long as the original antibody activity is not lost. Of various antibodies

 2

 It may be a derivative, recombinant or fragment modified into various forms known to those skilled in the art, including derivatives.

[0038] It is also possible to measure the expression level of the protein by binding a reporter gene encoding a labeling substance to the gene encoding the protein, expressing these fusion proteins, and measuring the labeled protein. It is. As an example of such a labeling substance, any protein known to those skilled in the art as a substance used for detection of the expressed protein can be used. Typical examples of such substances are GST, His, AviTag, V5 and FLA

 6

Various tag substances such as G, GFP (Green fluorescent protein), YFP (Yellow fluorescent protein), RFP (Red fluorescent protein) and BFP (Blue fluo) For example, proteins that emit autofluorescence in various colors in the visible light range such as green, yellow, red, and blue, or proteins that change in color over time. Furthermore, it is possible to use a protein that emits fluorescence from infrared light or ultraviolet light.

 [0039] In addition, if a protein having a specific antigenicity is used as a labeled protein and a fluorescent antibody that recognizes the antigenicity is used, it is possible to impart fluorescence to the protein to be measured. Furthermore, an enzyme protein such as luciferase can be used as the labeling protein, and a substrate that can produce a fluorescent substance by the action of the enzyme protein can be used.

 [0040] On the other hand, the expression level of the Fragl gene (mRNA or cDNA) is measured by various probes designed as appropriate based on the base sequence of the gene (DNA) encoding the protein described in the Examples of the present specification. Alternatively, it can be carried out by methods known to those skilled in the art such as Northern analysis, various quantitative PCR methods such as RT-PCR method, and microarray (DNA chip) method using primers.

 [0041] Further, in the detection method of the present invention, the DNA damage is caused by any measurement method / means known to those skilled in the art for the Fragl gene, the gene product or a phosphate thereof, or a cellular response involving them. It can be detected by measuring. These cell responses need not necessarily be quantitative, and the effects of the present invention can be sufficiently obtained even by qualitative or semi-quantitative measurements such as visual observation depending on specific measurement methods and means. I can do it.

 [0042] Specific examples of powerful cellular responses include apoptosis, activation of various caspase proteins such as Caspase 7, and molecules that induce apoptosis such as Bax, Fragl and Rad9 Reaction 'changes such as dissociation with Rad9 and binding of Rad9 and Bcl2. Apoptosis can be measured, for example, by measuring the number of cultured cells by any method known to those skilled in the art and determining its viability. The reaction 'changes such as activation of various molecules or dissociation or binding of proteins, for example, as described above, methods using various immunological specific reactions using antibodies against proteins involved in these reaction' changes Can be measured. Such various antibodies can also be easily produced according to the above-described method for producing anti-Fmgl antibody.

[0043] In the detection method of the present invention, examples of the subject include animal cells such as humans and mice. Any cultured cell line known to those skilled in the art derived from or cancer cells derived from excised tissues such as cancer tissues and other various cells can be used.

 [0044] Furthermore, a transformed cell in which Fmgl gene expression is inhibited or suppressed, or a transformed cell into which a gene encoding Fmgl, a mutant thereof, or a part thereof is introduced can be used. There are no particular restrictions on the host cells used to produce such transformed cells, but it is usually preferable to use the same type of cells as the original source of the Fragl gene. Such transformed cells can be easily prepared according to the method already described.

 [0045] For example, a transformed cell in which Fmgl gene expression is inhibited or suppressed includes an Fmgl gene represented by antisense oligo DNA, antisense cDNA, siRNA, dsRNA that produces it, or ssRNA, or the like. Can be prepared by the antisense method or the RNAi method using a nucleic acid molecule that is hybridized under stringent conditions with a partial region or a complementary strand thereof. Such a nucleic acid molecule can be easily designed and prepared by those skilled in the art according to the base sequence information of the Fmgl gene disclosed in this specification.

 [0046] In addition, as an example of a variant of Fmgl, there is an amino acid mutation in a motif that is considered to be a phosphate 匕 site or an Rb binding site by Atr, as specifically described in the examples of the present specification. Protein variants that have lost their function as a result of being introduced can be mentioned.

 [0047] Further, as an analyte in the detection method of the present invention, the Fmgl gene is defective or Fragl gene obtained by genetic engineering operations, as specifically described in the examples of the present specification. Genetic modifications such as knockout or knockdown animals (except humans) whose expression is inhibited or suppressed, or transgenic animals (excluding humans) into which genes encoding Fmgl, its mutants or a part thereof are introduced It is possible to use cultured cell lines established from animals or their genetically modified animals.

[0048] As already described, DNA damage is caused by, for example, various carcinogenic substances such as various DNA replication inhibitors, carcinogens and potential carcinogens, and electromagnetic waves such as radiation and ultraviolet rays. And other exogenous carcinogenic stimuli such as fever, or endogenous factors (replication stress, etc.) Caused by. Therefore, the method for detecting DNA damage of the present invention comprises a screening method for an exogenous carcinogenic compound or an anticancer agent (including an antitumor agent, a carcinogenesis preventive agent, etc.), or a disease, cancer or cancer involving Fmgl expression or activity. It can be used for cancer cell testing or diagnostic methods. Indirect situational evidence or observational powers, such as Jenne, DE, Tinschert, S., Stegmann, E., Reima nn, H., Nurnberg, P., Horn, D., Naumann, I., Buske, A., Thiel, G. A common set of at least 11 functional genes is lost in the majority of NFl patients with gross deletio ns.Genomics. 66. (1 ), 93-7, 2000), such as diseases that cause genomic changes near the Fmgl locus of chromosome 17 where the Fmgl gene is located, for example, neurofibromatosis Can do.

[0049] For example, exogenous carcinogenic stimuli such as carcinogens can be screened in the following steps:

 (a) culturing the above-mentioned various transformants or cells (subject) in the presence of the test substance, or allowing the test substance to act on the genetically modified animal (subject) ,

(b) measuring the expression level of the Fragl gene, the production amount of the Fragl gene product or a phosphate thereof, or the cellular response induced by them in the subject, and

 (c) selecting a test substance that increases or decreases the expression level, the production level or the cellular response.

 [0050] Also, for example, anticancer agents can be screened in the following steps:

 (a) The various transformants or cells (subjects) shown above are cultured in the presence of the test substance under conditions that cause DNA damage, or the genetically modified animals (subjects) A step of allowing a test substance to act on (sample),

 (b) measuring the expression level of the Fragl gene, the production level of the Fragl gene product or its phosphate in the subject, or the cellular response induced by them, and

 (c) selecting a test substance that increases or decreases the expression level, the production level or the cellular response.

[0051] Here, for example, (1) a transformed cell in which Fmgl gene expression is inhibited or suppressed, (2) a transformation into which a gene encoding a mutant of Fmgl or a part thereof is introduced Cells, (3) knockout or knockdown animals in which expression of the Fragl gene is inhibited or suppressed, (4) transgenic animals into which a gene encoding a variant of Fragl or a part thereof is introduced (thereby (5) Overexpressed as a dominant negative of Fragl), and (5) the animal-powered cultured cell line is less susceptible to detection of cellular responses that are more sensitive to DNA damage. Even when V is used as a test substance at a concentration, it is possible to screen exogenous carcinogenic stimuli such as carcinogens with high sensitivity. Also, it becomes possible to screen anticancer agents in the presence of a lower degree of DNA damaging stimulus. In addition, this genetically modified animal should exhibit DNA damage or related points of action due to cell activity during any time after exposure, even for substances that do not cause DNA damage immediately. It is also possible to detect potential DNA damage-like activity by substances that cause more similar pathologies. The relevant points of action may be 100% -matching the effects of the direct DNA damage pathway, but in many cases 99% or less, preferably 80% or more, in some cases 50% or more, or even more. Even below, it is possible to show the effect of exposure over time of the physiological operation of the cell.

 [0052] In contrast, for example, (1) a transformed cell into which a gene encoding Fragl has been introduced

 (As a result, it is overexpressed), (2) a transgenic animal into which a gene encoding Fmgl has been introduced (so that it is overexpressed), and (3) Established cell lines are less sensitive to cellular responses to DNA damage (“threshold” is high) and do not immediately initiate cellular responses to DNA damage, Since it continues to survive without leading to apoptosis, substances that have a sporadic effect that cannot be detected suddenly, such as potential carcinogens, or multiple exposures with multiple types of analytes Effective screening is possible.

[0053] The kit used for the DNA damage detection method, screening method, or test or diagnosis method of the present invention can have an appropriate configuration according to the specific measurement principle of the method of the present invention. The kit is typified by, for example, an anti-Fragl antibody, an antibody against a protein involved in various biological responses induced by DNA damage, or an antisense oligo DNA, an antisense cDNA, or siRNA or a dsRNA or ssRNA that produces it. The Nucleic acid molecules that hybridize under stringent conditions with the Fragl gene or a part thereof, or a complementary strand thereof, primers for amplification of the above genes and hybridizers for measuring the expression level of the Fragl gene (mRNA or cDNA) It can contain a probe for the application. Each of these various primers and probes has an appropriate length, for example, 10 to 300 consecutive base sequences, depending on the application.

[0054] Various primers, probes, or antibodies included as components in the above kit are labeled with an appropriate labeling substance such as any radioactive substance, fluorescent substance, and dye known to those skilled in the art. May be. Furthermore, the above kit contains other elements or components known to those skilled in the art, for example, various reagents, enzymes, buffers, reaction plates (containers), etc., depending on the configuration's purpose of use.

 is there.

 Hereinafter, the present invention will be described in detail by way of examples. However, the technical scope of the present invention is not construed as being limited by the description of the following examples. Further, unless otherwise specified, the following examples are conventional methods in the art and standard methods known to those skilled in the art, such as bambroo and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold. Implemented according to genetic engineering and molecular biological techniques described in Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. did. In addition, the contents of the literature cited as a reference in this specification constitute a part of the disclosure content of this specification.

 Example 1

 [0056] (clone of mouse Fragl gene cDNA)

Pregnant mouse 13.5 days of fetal subcutaneous tissue was isolated, placed in Dulbecco's modified Sidar medium with 10% serum (10% FCS / DMEM), cultured in a plastic dish for 5 days, and then passaged for 2 passages. MEFs (mouse fetal fibroblasts) were prepared. In order to block the cell cycle in the G1 phase, 2xl0 ⁵ cells were cultured for 24 hours in 10% FCS / DMEM containing thymidine (2.5 mM) (referred to as primary block). Wash cells with phosphate buffered saline (PBS) and thymidine ヽ Freshly cultured in 10% FCS / DMEM for 10 hours, and again cultured in 10% FCS / D MEM containing thymidine (2.5 mM) for 16 hours (referred to as the secondary block. Primary and secondary blocks) Together called a double thymidine block;).

 [0057] This cell was used as a control, while as a replication-inhibited cell, affidecorin (0.4 ^ M; Sigma, St. Louis, MO) dissolved in DMSO (0.2%) during the secondary block was used as the cell culture medium. (Additional secondary block) and when replaced with fresh 10% FCS / DMEM without thymidine, add affidocholine (0.4 μΜ) and caffeine (0.2 μΜ) for 4 hours. Cultured and replication-inhibited loaded cells were prepared. Collect these cells, extract RNA according to the Qiagen kit and its instructions, and use 2 μg of that RNA to create a cDNA synthesis kit (Invitrogen) with Superscript II reverse transcriptase and oligo-dT or random primer and its CDNAs were synthesized according to the instructions. In order to identify cDNAs of genes that express differently, subtraction kit (the control cell is the tester, the replication-inhibited cell is the driver, and the tester power driver is subtracted by the noise visualization. Subtraction was performed according to Clontech) and its instructions. In addition, according to the instructions, after two rounds of hybridization, cDNAs were amplified, incorporated into a pcDNA vector (Clontech), and sequenced by a sequencing reaction.

 [0058] We analyzed 155 clones as a starting clone, but as a result of database search (http: 〃 www.ncbi.nlm.nih.gov/BLAST/), 86 clones (55%) were in a state where the known expressed sequence tags (ESTs) of the mouse matched or substantially matched in the computer database. Among them, 86 clones contain multiple fragments of different cDNA parts that are actually derived from the same gene, and two sequences were found in the greatest common divisor, Fragl / N and I named it Fragl / C.

[0059] As a result of database search (http: 〃 www.ncbi.nlm.nih.gov/BLAST/), Fragl / N and Frag 1 / C were confused until they were located on mouse chromosome 11. It is not possible to predict from the database search that Fragl / N and Fragl / C are the same gene. Therefore, we conducted a Northern plot experiment using Fragl / N and Fragl / C as probes (Fig. 1B), and found that both clones had very similar expression patterns and gene sizes. Based on Fragl / C sequence information After successful trial and error, we succeeded in synthesizing continuous cDNA connecting the 3 'end of Fragl / N and the 5' end of Fragl / C by PCR from a cDNA library. Fragl / N and Fragl / C are identical. Prove that it is in the gene. Table 1 below shows PCR polymerases used in the experiments. RTPCR—MmELGl—F1 and RTPCR—MmELGl—F2; RTPCR—MmELGl—R1 and RTPCR—MmELGl—R2; RTPCR—MmELGl—F3 and RTPCR—MmELGl—R4 were paired! / ヽ. When we look back at the database after crawling, the part connecting Fragl / N and Fragl / C sandwiched the intron and was equivalent to the part lacking the sequence from the mouse cDNA database. Full-length unknown function including C Frag (CtH8 / Rad 24 / Elgl—related gene 1) As a izs child to ~~ ta '~~~ (http://www.ncbi.nlm.nih.gov/) Registered (AY557610). In addition, the following SEQ ID Nos. Are shown in this specification: Mouse Frag 1 mRNA (SEQ. ID. NO: 1), Mouse Fragl ORF (SEQ. ID. NO: 2), Mouse Fragl protein (SEQ. ID. NO: 3).

[0060] [Table 1]

RTPCR- MmELGl- Fl: ACATCAGAAAAGCACAACCTGTATACAGCAG

 'RTPCR-MmELGl-F2: AGGACACTATTCCAGTGAAGGCTTCCACC

 RTPCR- MmELGl- Rl: TTTGCAGAACGTGCCTGGTGCCTAGAGGAC RTPCR-MmELGl-R2: TCTCTGGCGACTTCTCCTTTGCAGAACGTG RTPCR-MmELGl-F3: GTGATGAGTTTAGTCTTGAGAATAGAGAC RTPCR-MmTAGTCTCAGATCRTTC-TC4

 [0061] (Cloning of cDNA of human Fragl gene)

Based on the mouse Fragl gene cDNA, human database search (http: 〃 www.ncbi.nlm.nih.gov/BLAST/) results in known expressed sequence tags (ESTs) and computer There is a clone that matches or substantially matches in the database, and is the cDNA of the human Fragl gene (substantially matched is less than 100%, but over any region above 200 bp> 95% The cause of this partial discrepancy is caused by the error in the base sequence test on the database side and the error in the base sequence test on the user side, or This is due to the mixture of the two, but it is common for the screening stage to be the same in practice). Table 2 and Table 3 show the PCR primer combinations and base sequences of each PCR primer used in the cloning experiment of the human Fragl gene cDNA. The human FRAG1 gene thus identified was registered in a database (http: 〃 www.ncbi.nlm.nih.gov/) (AY557611). In addition, the following SEQ ID Nos. Are shown in this specification name: Human Fragl mRNA (SEQ. ID. NO: 4)), Human Fragl ORF (SEQ. ID. NO: 5), Human Fragl protein (SEQ. ID. NO: 6)).

[Table 2]

EL-F1-U1 and EL- Fl- Dl

 EL-F1-U1 and EL-F1-D2

 EL- F 卜 U2 and EL- Fl- Ul

 EL-F1- D2 and EL- Fl- D2

 EL-F2-U1 and EL-F2-D1

 EL-F2-U1 and EL-F2-D2

 EL-F2-U2 and EL-F2-Ul

 EL-F2-D2 and EL-F2 ^ D2

 EL-F3-Ul and EL-F3-Dl

 EL-F3- Ul and EL-F3-D2

 EL-F3- U2 and EL-F3-U1

 EL-F3- D2 and EL-F3- D2

 EL-F4-U1 and EL-F4-D1

 EL-F4-Ul and EL-F4-D2

 EL-F4-U2 and EL-F4-U1

 EL-F4-D2 and EL-F4- D2

 EL-FZ- Ul and EL-FZ-D1

 EL-FZ-U1 and EL-FZ-D2

 EL-FZ- U2 and EL-FZ- Ul and

EL-FZ-D2 and EL- FZ- D2 _C Layer VH Ring JMW

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 30VW3XIIVD9i) IDIII9II9I3DDXV

 03IIIV3I3WI39V1VIIII1I0WI9

 99I0V01X3WIVID0I1IV3I0WI ια- εί- Ί3

 OOXIIDIVWXOailOiJVOOOWDIOIV

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 3IV0VI9II0XVIIVI0VDIIDVIIX33 -Ζί-ΊΆ

 3IXID9IVW03IVI99I3IIWW90I Zil-Zl-ΊΆ

 9I3V0V0VIX9V0VIIIW099IVWW ΙίΙΙΉ

3VXW99I009IIIII91DI3X030II19

 3IWI3IVI9V33DD9VIWSD100DII ΐα-ΐί-Ί3

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 [ε 挲] Κ900]

800C0C / 900Zdf / X3d 93 6 .9060 / 900Ζ OAV [0064] (Mouse Fragl protein)

 In order to examine whether the mouse Fragl gene cDNA can be synthesized as a protein, an in vitro transcription / translation system using a rabbit reticulocyte extract was performed using a Promega kit. As a result, it was shown that the mouse Fragl gene cDNA can be synthesized as a protein (Fig. 4B). Furthermore, using a rabbit antibody polyclonal antibody (the antibody described in Example 11) that was prepared based on the amino acid sequences of mouse Fragl protein and human Fragl protein and recognizes a region common to both as an epitope. Western plots showed that mouse Fragl protein was expressed in the mouse cell extract (shown as α Fragl in FIGS. 2, 3, 4, 5 and 6).

 Example 4

[0065] (Human Fragl protein)

 To examine whether mouse Fragl gene cDNA can be synthesized as a protein, a Promega kit was used as an in vitro transcription / translation system using a rabbit reticulocyte extract. As a result, the cDNA of the human Fragl gene can be synthesized as a protein. When Western plotting was performed using the Usagi polyclonal antibody (produced in Example 11), the human Fragl protein was expressed in the human cell extract. (Fig. 12

) o

 Example 5

[0066] (Change in expression of Fragl)

Since the FRAG1 gene was cloned using the expression change in response to replication inhibition as an index, the gene groups related to replication so far (RFC1, RAD17 and CTF18) were also combined. For Fragl, Fragl / N, Fragl / C As a probe, a Northern analysis was performed for comparison. For Northern analyses, MEFs with synchronized cell cycle were used to expose affe decoline (APD) or DNA alkylating agent methyl methane sulfonate (MMS). Phyadecolin is an inhibitor of polymerase alpha and MMS induces a delay in the replication fork, both of which have specific action points, but both result in inhibition of the replication process. Two were used to investigate whether the observed changes in Fragl were due to a specific point of action or to a phenomenon associated with regular replication inhibition. As a result, Both exposure to Fragl / N and Fragl / C were similarly markedly reduced by choline or MMS exposure. On the other hand, this change was not so noticeable in RFC1, Radl7 and CTF18 (Figure 1A).

 [0067] Furthermore, in order to determine whether the mechanism by which the transcript of Fragl decreases after inhibition of replication is due to reduced transcription or increased degradation, actinomycin D suppresses nascent RNA synthesis, The stability of the transcript was examined over time with and without affedecolin. As a result, when nascent RNA synthesis was suppressed and alphadecolin was added, the half-life of Fragl mRNA was 4 hours or less, while the half-life of CTF18, RFC and RAD17 mRNAs was 15 hours or more. This difference decreased if no affeidolin was added. Therefore, it was found that the stability of transcripts under replication inhibition was specifically decreased by Fragl (Fig. 7D and Fig. 7E). The base sequences of the primers used in PCR in the above experiments are shown in Table 4 below.

 [0068] [Table 4]

RFC: 5 '-GGGAGCTAATCAAGATGTCAGACAGG-3' &

 5 '-GTGCAAGGATAGGTCCTGAACAATCC-3';

RAD17: 5 '-AGATTCAAGCTATTGGTGGAAAAGATG-3' &

 5'-AAGATATGGCAAGAGCTGAGTCTGGAG-3 ';

CTF18: 5 '-CTCTGTGAGAAGACCGACAATGACATCC-3' &

 5 '-TGGCTGGAGGCAAAGAGCACGTGGAAGG-3'; and

AC IN: 5 '-ATTGAACATGGCATTGTTACCAACTGG-3' &

 5 '-GGCCATCTCCTGCTCGAAGTCTAGAG-3' Example 6

[0069] (Fragl replication inhibition sensitivity control)

In order to examine the biological effects resulting from decreased expression of Fragl, we tried to suppress endogenous Fragl expression using siRNA. Preparation of siRNA expression vector and introduction into cells was performed using Takara Kit and following the instructions. The siRNA shown in Table 5 below was introduced into the cells, and after RT-PCR and immunoblot analysis, the siRNA vector was transferred and purified. When selected with 10% FCS / DMEM containing a bite-mycin selective antibiotic (1 _μg / ml), the siRNA functioned effectively and markedly increased Fragl expression. It was confirmed that it was suppressed (Fig. 2A). The degree of suppression is ideally 90% or more, often 80% or more, and even 50% or more may represent an effective trait.

 [0070] Next, using these cells, MMS was added to the medium or irradiation with gamma-irradiation was performed. Here, MMS induces delayed damage of single-stranded DNA forks, whereas gamma-irradiat ion irradiation induces double-stranded DNA breakage damage. Two independent FRAG1 siRNA transfectants were examined, both of which were elevated compared to the sensitivity controls for replication stress (by MMS), and this change was associated with MEFs that synchronized the cell cycle to S phase. This is even more pronounced, suggesting that these forces also activate S-phase checkpoints. This change was more remarkable than the experiment with gamma-irradiation irradiation. Thus, decreased Fragl expression was found to enhance sensitivity to MMS (due to delayed damage of single-stranded DNA forks) (Figure 2B).

 [0071] [Table 5]

Example 7 [0072] (Activation of apoptotic pathway by inhibition of Fragl replication)

 When immunoblotting analysis of the Fragl protein was performed, it was observed that the Fragl protein expression decreased 2-6 hours after exposure to alpha-choline or MMS, and this decrease is known to be a homeostatic expression used as a control. The cytoskeletal actin or Radl7, which has been identified, was immediately activated (Fig. 3A). In other words, in the experimental system that can confirm the homeostasis of actin, it is degraded faster than Radl7, which means that Fragl has a function closely related to Radl7! And, uh, suggesting that Fragl has inherent properties! /

[0073] Further, when the expression of Bax protein was examined, in cells in which the cell cycle was synchronized with a double thymidine block and Fragl was suppressed by siRNA, 8 hours after release from GlZS, the gel was analyzed in an immunoblot analysis. It is observed that the protein fraction that migrates slowly in, which shows the activity of Bax (Fig. 3B). This change is highly universal because it was seen in two cells that were tested independently. Furthermore, this Bax activity phenomenon was observed after the MMS treatment in cells that had been suppressed for Fragl by siRNA, and fractionation of all time examined after cell cycle synchronization (mid-S phase) by double thymidine block. Observed in. In contrast, Bax activity was not observed in the cells of the control siRNA experiment (Fig. 3B). This result shows that Fragl suppression induces the activity of Bax, one of the molecules that induce apoptosis, and that the induction of Bax activity is significantly enhanced by MMS treatment. Shows increased sensitivity to In addition, it was observed that p53 serine 15 was phosphorylated after MMS exposure. This was observed in two independent cells (# 403 and # 406) in common and was highly woven. (Figure 3B). In addition, even in the absence of MMS loading, p53 serine 15 phosphate was observed in Fragl-suppressed cells with siRNA synchronized in the cell cycle after 4 or 8 hours. This means that even if MMS is not loaded, if Fragl is suppressed strongly, it is also detected by the genomic damage sensor of the p53 pathway, and the life response system is not networked. It shows that. Compared to p53, Fragl behaves differently, indicating that it has a different function with crosstalk as a system. On the other hand, in the control siRNA experiment, phosphorylation of serine 15 of p53 was observed only after MMS exposure, and Fragl suppression was observed in cells without MMS exposure. Reduced Fragl because the Bax activity was stimulated 4 or 8 hours after release of the synchronized cells (0,4,8 hours without MMS exposure with FraglsiRNA in Fig. 3B) Increases the sensitivity of the response to genomic toxins (replication stress). The changes in Mdm2 were not obvious, suggesting that Mdm2 itself is not close to the Fragl pathway.

On the other hand, activation of caspase 7 was observed in cells in which Fragl was suppressed by siRNA, which was specific to Fragl suppression since it was not observed in control siRNA experiments (FIG. 8A). Moreover, it was observed that when Fragl was suppressed by siRNA or exposed to MMS, cytochrome c release from mitochondria was promoted (FIG. 8C).

 Together, the above facts suggest that Fragl suppression increases cellular susceptibility to DNA damage and activates Bax to induce apoptosis through the pathway.

 [0075] Furthermore, in order to investigate whether p53 is involved in the activation of Bax in cells that suppress Fragl by siRNA, MEFs (Trp53 deficient MEFs) in which p53 was genetically deleted were prepared and tested. As a result, the induction of Bax by suppressing Fragl by siRNA resulted in Trp53 (+/-) cells that had only one half of the p53 gene and lacked one of the two alleles of the p53 gene, 2 It was found that Bax was similarly induced in Trp53 (-/-), which lacks both alleles and has no p53 gene genetically (Fig. 8B). In addition, when we examined whether serine 15 of p53 is phosphorylated, it was found that Trp53 (+/-) cells were phosphorylated and Trp53 (-/-) was phosphorylated. It was confirmed. These results indicate that Bax is activated independently of the state of p53, and that Bax activity after replication inhibition in the Fragl pathway is not related to p53. Nikkori p53 was different from that of p53.

 Example 8

[0076] (Fragl and Rad9-Bcl2 pathway)

As previously known, the Rad9 molecule is known to play a role in inducing cell death following DNA damage, which interacts directly with the anti-apoptotic molecule Bcl2. By suppressing. Therefore, in order to investigate the Fragl signaling pathway, we co-immunoprecipitated interactions with proteins that play a role after replication inhibition. It was examined by the downstream analysis (Fig. 9A). After trial and error, immunoprecipitation using an anti-Rad9 antibody followed by an immunoblot using an anti-Fragl antibody revealed binding between the two. It was observed that Fragl expression was reduced after exposure to affedecolin or MMS, with a concomitant decrease in the binding of Fragl to Rad9 (Fig. 4A). Instead, Rad9 binds strongly to Bcl2. That is, when replication is inhibited, the Fragl-Rad9 bond dissociates with time, and a Rad9-Bcl2 bond is generated instead. In the former Fragl-Rad9 bond dissociation, the above-mentioned decrease in Fragl expression was involved, and as described later, it was found that Fragl phosphorylation modification and Rb binding modification were involved. It is suggested that it is precisely controlled by.

[0077] A recombinant dartathione S transferase (GST) -Rad9 fusion protein was used, and the binding site was examined in detail by biochemical pull-down assay. When GST binding Rad9 and a short fragment in which Fragl was artificially created in various parts by in vitro transcription and translation reaction were used, a binding experiment was conducted. GST binding Rad9 bound to the F2 fragment of Fragl. It did not bind (Figure 1C, Figure 4B). This suggests that the F2 fragment part binds to Rad9 and controls cell death. A gene transferer of ME F that expresses a specific fragment of Fragl was prepared and tested for colony-forming ability. When the F2 fragment of Fragl is overexpressed, colony-forming ability after genome poisoning is relatively high. (Fig. 4C), suggesting that the F2 fragment is involved in cell death control, resulting in changes in colony formation, and that it is also involved in cell death control by binding to Rad9. Was

[0078] Further experiments were carried out using this F2 fragment gene transfectant (Fig. 4D). Here, F2 fragment gene-introduced body in which cell death was suppressed was introduced, and Rad9 was further introduced, and cells containing the gene were selected using hygromydn resistance as an index. As a result, the result of overexpression of Rad9 over the F2 fragment gene transducer induced apoptosis. On the other hand, when Rad9 mutant Rad9Ndel lacking the binding part to Bcl2 was introduced instead, apoptosis was suppressed. This is probably because Rad9 mutants cannot bind to Bcl2 and therefore cannot induce apoptosis.

[0079] Furthermore, when Bcl2 was introduced into the F2 fragment gene transducer instead, apoptosis was evident. It was markedly suppressed (Fig. 4D). For confirmation, even when observed with a confocal microscope, the F2 fragment gene-transfectant suppressed cytochrome c release after MMS exposure (Figure 9B).

[0080] These results strongly suggest that Fragl regulates the binding of Rad9 and Bcl2 and induces apoptosis induced by DNA damage. In this specification, each fragment of Fragl is represented by the following SEQ ID NO: Human Fragl Fl fragment (SEQ. ID. NO: 7), Human Fragl F 2 fragment (SEQ. ID. NO: 8), Human Fragl F3 fragment (SEQ. ID. NO: 9), Human Fragl F4 fragment (SEQ. ID. NO: 10), Human Fragl FZ fragment (SEQ. ID. NO: 11).

 Example 9

 [0081] (In the cell cycle mid-S phase, Fragl controls a novel signaling pathway between Atr and Rad9.)

 In order to investigate the function of Fragl, it was examined whether or not the binding of Rad9 and Fragl was dependent on the cell cycle after exposure for 0.01% MMS for the time indicated (upper figure 5A). As a result of immunoprecipitation using anti-Rad9 and anti-Fragl antibodies, the binding of Rad9 and Fragl was found to be weak in G1 phase cells synchronized with the double thymidine block, and then in S phase. When I got it, I got stronger. Similarly, after MMS exposure, the binding between Rad9 and Fragl decreased, and instead the binding between Rad9 and Bcl2 was promoted (Fig. 5A bottom). This result shows that Fragl enhances the sensitivity of Rad9 to genomic venom stress in the S phase of the cell cycle, and is consistent with the above data.

[0082] When observed with a confocal microscope using anti-Fragl and anti-Atr antibodies (Figure 10), Fragl and Atr coexist 8-12 hours after MMS exposure, and Fragl forms a focus before Atr. It is observed that the Rad9 pathway is involved in the Rad9 pathway, suggesting that Fragl functions via the Atr kinase response to DNA damage. Therefore, Fragl was shown to control a new signaling pathway between Atr and Rad9

[0083] Furthermore, the present inventor, after trial and error using a database with other proteins (http: 〃 www.ncbi.nlm.nih.gov/), shows Fragl as shown in FIG. 1C. , Which contains a motif that suggests a binding site between the phosphorylation site by Atr kinase and the tumor suppressor gene product Rb, Since it has been suggested that biochemical modifications are at least partially involved, these

We decided to investigate whether the interaction of Atr kinase R with Rb modifies the novel signaling pathway of Fragl. In order to examine the importance of the motifs found above, various mutants were created. Ser ¹¹⁶⁹ and Ser ¹¹⁸⁷ were introduced by introducing a point mutation by converting the nucleotide sequence encoding the phosphorylated amino acid (serine) of Atr kinase into a similar amino acid (alanine) that cannot be phosphorylated. Each was converted to alanine Ala. Similarly, in order to convert the binding motif of Rb into a motif in which Rb cannot bind, LxCxE ¹⁴³² was converted to create a full-length sequence containing LxGxK ¹⁴³² or LxGxE ¹⁴³² . (Here, why LxGxK ¹⁴³² or created a LxGxE ¹⁴³² and two variants that use both converted one or two amino acids from the wild-type experiments, respectively, is added a more detailed discussion ) As a result of immunoprecipitation, it was confirmed that wild-type Fragl does not bind to the Rb-binding force of LxGxE and LxGxK mutants. In addition, the binding between wild-type Fragl and Rb was more apparent in the G1 phase than in S in cells with synchronized cell collection cycles (FIG. 5B). After exposure for the indicated time of 0.01% MMS, the expression of wild-type Fragl was reduced to less than the sensitivity as a result of Western blotting. Fragment of LxGxK mutant Fragl was detected without loss, but weakly LxGxE. Similar results were observed. Overall, Fragl controls the novel signaling pathway between Atr and Rad9 in the mid-S phase biological response to replication inhibition, and as a result, it is clear that Fragl has a function to increase susceptibility to genomic toxins. It was.

To investigate further, we used TransIT-TKO transfection reagent (Mirus, Madison, WI) as described in the instructions to introduce endogenous Atr siRNA into cells and suppress endogenous Atr (Figure 5C). ). Western blot results showed a decrease in endogenous Fragl in control cells after 0.01% MMS exposure for the time indicated, whereas suppression of Atr by siRNA was endogenous to MMS exposure. Inhibited the reduction of Fragl. As a result of immunoprecipitation, binding of Rad9 and Fragl was also significantly reduced when Atr was suppressed after exposure for 0.01% MMS. The decrease was observed more clearly in cells in which ATR was suppressed by siRNA than in control cells. Therefore, from the above, Atr has a new function, which is related to the control in two stages. It is powerful. One is the stage where Rad9 binds to Fragl, and the other is the stage where the amount of Fragl decreases in response to DNA damage.

 Example 10

 [0085] (In the cell cycle mid-S phase, Fragl regulates apoptosis through both Atr phosphorylation and Rb binding)

 We investigated whether the binding and phosphorylation of Fragl and Rb is related to the bond dissociation of Rad9 and Fragl. To that end, we created stable gene transfectants that express wild-type Fragl and mutant Fragl, exposed them to MMS, and analyzed the proteins (Figure 6A). After exposure to G1 phase cells with 0.01% MMS, synchronized with the cell cycle with double thymidine block, binding between Fragl and Rad9 decreased after 4 and 8 hours after resuming the cell cycle. The decrease was suppressed in the Serll69Ala mutant and LxGxK mutant, and the decrease was suppressed in the Serll87Ala mutant and LxGxE mutant although it was weak. These results suggest that Atr-induced phosphorylation promotes Rad9 dissociation, and that Rb and Fragl binding are also directly or indirectly involved in this activity of Rad9. Furthermore, looking at cells that have undergone apoptosis, the Serll69Ala mutant and the LxGxK mutant, among the mutants, show the (resistant) traits that are less prone to apoptosis even if DNA damage occurs ( Figure 6B). This result further reinforces the importance of Fragl-Rad9 binding in cell death induction. Lastly, we studied wild-type ATR (ATR-wt) or kinase-dead ATR (ATR-kd) with Fragl by transfecting cells with GibcoBRL gene transfer reagents according to the instructions for use. As a result, after exposure for the time indicated 0.01% MMS, the decrease in Fragl expression was not suppressed by ATR-wt, which was suppressed by ATR-kd. Thus, the conclusion was reconfirmed that Atr's phosphate chain plays a role in the DNA damage response controlled by Fragl-Rad9 (Figure 11).

 Example 11

 [0086] (Antibodies against Fragl and related reagents)

In order to elucidate the properties and functions of Fragl, a part of the amino acid sequence of mouse Fragl 345 CS LSDPENEQPVQKRKSN 362 was synthesized and KSL adjuvant was immunized, then the rabbit was immunized, and serum was collected. Use affinity purification on part of the sequence Here, a polyclonal antibody was prepared according to a conventional method. Examples of the use of this antibody were as described above, and demonstrated the function of Fragl in the DNA damage response (Figs. 2, 3, 4, 5, 6, 8, 9, 10, 12). Immunization of all or part of the Fragl protein artificially modified with phosphorylation (CGR QIL <pS> QLKEA, which is part of the antigen sequence of the phosphorylated antibody against <1169S> of human-fragmented biosynthesis) FIG. 12 shows an example of an anti-phosphate Fragl antibody prepared by using as a source. This antibody can also be used for mouse Fragl.

[0087] The primary antibodies used in the above examples of the present invention were the following commercially available products. human p53 (Cell Signaling, a component of kit # 9919), pno sphorylated p53 (Ser 15) (Cell Signaling, a component of kit # 9919), Mdm2 (Santa Cruz Biotechnology, # 5304), Rb (BD Biosciences, # G3- 245), Atr (EMD Biosciences, San Diego, CA, # ab-2), Rad9 (Santa Cruz Biotechnology, # 8324), phospho- H2AX (Upstate, Chicago, IL, # 07—164), mitochondria hemicon, Temecula, CA, #RbtX, A b3598), Orc2 (EMD Biosciences, # NA73), Rad9 (Santa Cruz Biotechnology, # 10465) and Atr (Santa Cruz Biotechnology, sc-1887), cytochrome c (BD Pharming en , San Diego, CA, # 6H2.B4, 556432), phospho— H2AX (Upstate, Chicago, IL, # 05—636), Grb2 (BD Transduction Laboratory, # 610111), V5 (Invitrogen, # 46-705), Flag (Sigma-Aldrich, St. Louis, MO, # M-2), and actin (ICN, Irvine CA, # C4).

 Example 12

[0088] (Generation of Fragl-deficient mice)

In the case of a Fragl-deficient mouse (knockout mouse), first, the genomic region of the mouse Fragl gene is subcloned from BAC (E. coli artificial chromosome), and the vector shown in FIG. 13 is prepared by gene recombination technology. Gene knockout methods include, for example, using appropriate drugs (eg, tetracycline 'steroid derivatives' zinc derivatives), additional genetic manipulation (eg, LOXP sequences), or to track expression in the mouse being produced. Any method known to those skilled in the art, such as additional insertion or replacement of one of these (for example, green fluorescent dye (GFP)), can be mentioned. Using this knockout vector, for example, stem cells of rodents such as mice and rats (for example, embryonic stem cells 'germline stem cells / embryonic tumor stem cells' embryonic teratoma stem cells are introduced into the This recombination is carried out to produce cells having the Fragl gene or an allele modified at the Fragl locus. These cells are introduced into early embryos (eg, the blastocyst stage is 4 or 8 cell stage) and placed in the uterus of pseudopregnant mice to give birth. After this, it is possible to create a Fragl-deficient mouse by proceeding with the breeding of chimeric mice by using a general method.

[0089] Furthermore, as an application example, a conditional knockout that artificially controls knockout by using a specific gene sequence prepared in advance of gene recombination and expression (for example, Cre-LoxP sequence system) ) Or an expression induction system that artificially controls the expression of the target gene (examples include, but are not limited to, methods using insect-derived hormones such as tetracycline, zinc, steroids, corticosteroids, and etadyson) )

[0090] Furthermore, the Fragl gene modification is performed by crossing the knockout animal thus created with other genetically modified animals (for example, animals incorporating a fluorescent dye gene, other tumor suppressor genes, or genetically engineered cancer-related genes). It is also possible to create hybrids that enhance and diminish the effect of.

 Example 13

 [0091] (Create Fragl Transgenic Mouse)

In the case of a Fragl transgenic mouse, for example, the mouse Fragl gene is incorporated downstream of the promoter to produce a vector (FIG. 14). The full-length cDNA of the Fragl gene, the full-length cDNA corresponding to the Phosphate-deficient Fragl created by mutating the base corresponding to the amino acid of the Phosphate-site, and a part of the Fragl gene Furthermore, it is possible to use the full length or a part of the DNA produced by the base insertion / substitution 'deletion' insertion 'fusion or the like to the Fragl gene. Representative examples of promoters include CAG and CMV promoters, and C-KIT and TEC promoters as tissue-specific promoters. Inducible promoters that respond to appropriate drugs such as tetracycline 'steroid derivatives' zinc derivatives can also be used. In addition, sequence elements for the purpose of additional genetic manipulation to make Fragl expression “increase / decrease” unchanged (for example, LOXP sequence), and markers for tracking the expression in the mouse to be produced (for example, Green fluorescent dye (GFP)) can be additionally inserted or replaced. Thus The prepared vector is injected into fertilized eggs of rodent experimental animals such as mice and rats, and placed in the uterus of pseudopregnant mice to give birth. After this, it is possible to create a transgenic mouse by proceeding with the mating of the transgenic mouse according to a general method. In addition, a cross between the transgenic animal thus created and another genetically modified animal can be used to produce a hybrid that enhances, attenuates, or does not change the effect of the Fragl gene modification.

 Example 14

 [0092] B6 wild-type mice or Fhit +/- p53 +/- mice were orally administered with the carcinogen NMBA (N-methylbenzylamine) 2 mgZg twice a week, 6 times a week. After 10 days, 37 days, 58 days and 84 days, the dissected tissues were analyzed. The number of tumors formed in the front stomach was counted, and the frequency of mice with tumors of 2 mm or more was shown as the tumor frequency. The tissue was fixed in formalin, embedded in norafin, and a tissue section was prepared. After blocking with 2.5% BSA and 2.5% serum, staining with anti-phosphorylated Fragl antibody, anti-Fhit antibody, and anti-p53 antibody, staining with secondary antibody, color development detection with DAB, and counterstaining of nuclei were performed. In addition, adjacent sections were stained with H & E. The force of H & E staining specimens was also summarized in Table 6 by distinguishing tumors into hyperplasias (H) and carcinomas (C) under a microscope. In addition, immunohistochemical staining revealed that the mass of the phosphorylated Fragl was increased by 50% or more during 20 visual field observations, and Fhit expression was 50% or higher during 20 visual field observations in comparison with normal tissues. Table 6 summarizes the number of cases that were attenuated or deficient in, and the number of cases in which p53 expression was increased, attenuated, or deficient in 50% or more of 20 observations. FIG. 15 shows that the tumor frequency increases with the number of days elapsed, and the tumor frequency in which increased expression of phosphorylated Fragl is observed. The numbers shown in the figure are the number of mice used in the experiment. FIG. 16 shows the tumor frequency after 84 days and the tumor frequency at which a marked change in the expression of each of the phosphorylate Fragl, Fhit, and p53 genes was observed.

[0093] Anti-phosphate Fragl antibody positive cases were as positive as immunohistochemical staining with anti-Fragl antibody. Anti-phosphate Fragl antibody and anti-Fragl antibody are considered to be useful. However, the results of the above-mentioned basic mechanism and the clearness of the immunohistochemical staining results are based on anti-phosphate The data were collected from antibodies. Fhit +/- p53 + / _ mice lack a single allele of a tumor suppressor gene and are highly sensitive to carcinogenic stimuli. 2 for Fhit +/- p53 +/- mice It is an animal model corresponding to a precancerous lesion lacking one allele of each of the two tumor suppressor genes. In this precancerous lesion animal model, Fragl is more highly phosphorylated at the phosphate site than the wild type. It was shown that In addition, the phosphorylation of this Fragl is caused by Fhit +/- p53 +/- mice with Fhit and p53 remaining deleted or mutated by inactivation of the environmental carcinogen NMBA. Prior to the progression of the multi-stage carcinogenic process of complete dysfunction, the carcinogenic process was detected at a high rate or with high sensitivity, indicating its usefulness as a very early carcinogenic marker.

[Table 6]

Industrial applicability [0095] The field of application of the method of the present invention is diverse, and representative examples thereof include carcinogenic susceptibility tests, carcinogenic safety tests, safety tests for potential genomic toxins, and carcinogenic substances that do not have direct genome toxicity. Examples include safety studies, carcinogenic pathology research, animal experiments for detection of early stages of carcinogenesis, DNA damage response pathology research, clinical tumor pathology research, and animal experiments for early detection of clinical tumors.

 [0096] In addition, anticancer agents are generally cytotoxins, and are used for cancer treatment by utilizing the difference in sensitivity between cancer cells and normal cells. The wider the difference in sensitivity, the more adverse reactions occur. << Anti-cancer drugs that can be used relatively safely. On the other hand, those with a narrow sensitivity difference range are anticancer agents that exert an antitumor effect on cancer cells, but may also affect normal cells at the same time.

 Even if the range of sensitivity difference is narrow, not only anticancer drugs but also widespread anticancer drugs, there is rather no confirmation that anticancer drugs will have no effect on normal cells until they reach the molecular cell biological level. It is undeniable that it causes potential cell damage to normal cells and has a spontaneous toxic effect! /.

[0097] As a result of the advancement of cancer treatment in the future and the improvement in survival rate, sporadic disorder caused by vigorous toxicity to normal cells, which has been apparent for early death, will become a problem in the future. There is a possibility of coming. In particular, as observed in experiments in vitro, at least a part of the activity can be detected even in animal carcinogenic systems using rodents, but anticancer agents that induce damage in the cell cycle S phase are cancer cells. At the same time, normal cells also induce DNA damage in the S phase, but the repair system has become abnormal in cancer cells (resulting from high levels of mutation accumulation due to the multi-stage carcinogenic process). The ability to survive and cell death In normal cells, the repair system functions to repair DNA damage and continue to survive.

[0098] However, it is unclear whether the repair of DNA damage in normal cells is completely repaired for all human genomes of about 3 billion base pairs. It is undeniable that the cells can survive as normal cells or non-cancerous cells. Since such cells are presumed to have at least partially damaged genomes, they can reach cancer after a period of time. Realistically, It is known that there are cases of spontaneous secondary cancer using anticancer drugs that act clinically during stage s (for example, but not limited to etoposide) for the treatment of malignant tumors, In view of this, detailed elucidation of the effects of anticancer drugs on the fidelity of the genome of normal cells is an extremely important issue, and it is a medical problem or lifeline based on the concept of controlling secondary carcinogenesis. The possibility of the development of science-related markets is strongly suggested.

Therefore, by detecting the degree of DNA damage when normal cells are exposed to an anticancer agent within the inspector using the method of the present invention, Prediction, 2) the optimal concentration or dosage to obtain the maximum anticancer effect in a form that minimizes DNA damage in normal cells of the anticancer drug, and 3) recent information on individual genomic information Combined with the prediction of side effects of anti-cancer drugs based on notable genetic diversity or individual differences (for example, but not limited to single nucleotide polymorphisms, polymorphism, etc.), more accurate information on genomic damage Various methods aiming at short-term side effect prediction (sensitivity related to overreaction or acute death) or long-term side effect prediction (risk of secondary cancer), etc., are possible.

Claims

The scope of the claims  [I] A method for detecting DNA damage, which uses, as a marker, an Fmgl gene in the subject, the gene product or a phosphate thereof, or a cellular response involving them.  [2] The method for detecting DNA damage according to claim 1, comprising measuring the expression level of the Fmgl gene.  [3] The method for detecting DNA damage according to claim 1, comprising measuring the production amount of the Fmgl gene product or its phosphate. [4] The method for detecting DNA damage according to [3], comprising measuring the production amount of the Fragl gene product or a phosphate thereof using an anti-Fragl protein antibody. 5. The method for detecting DNA damage according to claim 1, wherein the cellular response is apoptosis. 6. The method for detecting DNA damage according to claim 1, wherein the cellular response is activation of a molecule that induces apoptosis.  [7] The method for detecting DNA damage according to claim 6, which is an activated molecular force caspase protein.  8. The method for detecting DNA damage according to claim 6, wherein the molecule to be activated is Bax. [9] The subject is characterized by using a transformed cell in which Fmgl gene expression is inhibited or suppressed, or a transformed cell into which a gene encoding Fr agl, a mutant thereof or a part thereof is introduced. The method for detecting DNA damage according to claim 1. [10] The method for detecting DNA damage according to claim 9, wherein a transformed cell in which expression of the Fragl gene is suppressed by siRNA is used.  [II] As a test subject, knockout or knockdown animals (except humans), in which Fmgl gene expression is inhibited or suppressed, obtained by genetic engineering operations, or Fragl, its mutants or some of them The method for detecting DNA damage according to any one of claims 1 to 8, wherein the method is performed using a transgenic animal (excluding humans) into which a gene to be encoded has been introduced.  12. The method for detecting DNA damage according to claim 11, wherein the animal belongs to a rodent.  [13] The method for detecting DNA damage according to any one of claims 1 to 12, wherein the DNA damage is caused by exogenous carcinogenic stimulation. [14] The DNA loss according to claim 13, wherein the exogenous carcinogenic stimulus is due to a compound. How to detect scratches.  [15] The method for detecting DNA damage according to any one of [1] to [12], wherein the DNA damage is caused by cancer cells. [16] A screening method for an exogenous carcinogenic stimulus or an anticancer agent, which uses the method for detecting DNA damage according to claim 14.  [17] A method for testing or diagnosing a disease associated with Fmgl expression or activity, which uses the method for detecting DNA damage according to claim 15. [18] A method for examining or diagnosing cancer or cancer cells using the method for detecting DNA damage according to claim 15.  [19] A kit for use in the method for detecting DNA damage according to any one of claims 1 to 14, the screening method according to claim 16, or the test or diagnosis method according to claim 16 or 17. [20] An antibody that specifically recognizes the Fmgl gene product, its phosphate, or a partial polypeptide thereof.  [21] A nucleic acid molecule that is hybridized under stringent conditions with the Fmgl gene or a part thereof, or a complementary strand thereof. [22] Antisense oligo DNA, antisense cDNA, or siRNA or ds that produces it The nucleic acid molecule according to claim 21, which is RNA or ssRNA. [23] A knockout or knockdown animal obtained by genetic engineering manipulation that is deficient in the Fmgl gene or whose Fmgl gene expression is inhibited or suppressed and can exhibit an elevated cell response to DNA damage (human Or cultured cell lines established therefrom. [24] Transgenic animals (excluding humans) or cultured cell lines established therefrom, in which a gene encoding a Fmgl mutant or a part thereof is introduced to change the cellular response to DNA damage.