KR101098940B1 - MOESIN Serving as Biomarkers for Carcinogens - Google Patents

Abstract

The present invention provides a method for detecting a carcinogen, comprising the steps of: (a) contacting a test substance with a cell comprising a membrane-organizing extension spike protein (MOESIN) gene; And (b) measuring the expression level of MOESIN protein in the cell; An increase in the expression of the protein is determined to be a carcinogen; And it relates to a kit for detecting carcinogens. MOESIN biomarker discovered in the present invention shows very good surface efficiency for carcinogens. The present invention can be used to determine the carcinogenic potential of certain substances with improved accuracy. In addition, the present invention can be used for safety evaluation during drug development.

MOESIN, carcinogen, marker, genotoxicity

Description

MOESIN Serving as Biomarkers for Carcinogens

The present invention relates to a method for detecting a carcinogen and a kit for detecting a carcinogen.

Genome and proteomics research began to evolve after the completion of the Genome Project in 2000. In the United States, the National Center for Toxicogenomics was founded in September 2000 to conduct genome and proteomics research using microarray technology. Genome and proteomics research has been applied to almost all disease research, and is actively being studied in Korea. However, genome and proteomics research on general diseases is very inferior to the amount of research funding in developed countries and the number of researchers. Korea's protein-related research activities have been conducted since the early 2000s in line with the global trend, but the technology has reached a certain level, but the fields of toxic genomes, toxic proteomic toxicity, and toxicological information are showing wide gaps compared to foreign countries. To solve these problems, the Genome and Proteome Environmental Toxicology Center was established in November 2003. The Korean Society of Toxic Genetic Proteins was founded in November 2004 to establish an international safety assessment system to evaluate toxicity. Institutional competitiveness is also strengthening.

Recently, several pharmaceutical companies have been conducting research on ADME / Tox (Absorption, Distribution, Metabolism, Excretion, Toxicity) for new drug development research, and it seems that ADME / Tox prediction using proteomics will greatly contribute to cost reduction. have. Since December 2004, the Japan Institute of Industrial Technology has developed protein chips with 10,000 arrays and used them for drug toxicity and safety evaluation, including allergy diagnosis. Unlike animal experiments, the assessment of toxic exposures is very important when assessing toxic effects in humans. Exposure assessment studies are developing around biomarkers and genetic diversity studies, and the importance of exposure assessment specialists is growing. In particular, humans need to have a quantitative evaluation of various lifestyles because they affect both toxic exposure and health effects.

With the development of molecular biology, genome and proteomics research is a global trend, and the application of toxic genomics and toxic proteomics applied to toxicology is not only basic research field, but also new drug development, preventive medicine, food safety, environmental risk, forensics, etc. It is attracting attention as an application and prediction technology that can be used in various fields of real life. In Korea, research on toxic genomics and toxic proteomics has been conducted in hazard assessment and toxicity assessment, but the establishment of a database utilizing proteomics is still insufficient compared to international technological advances. The development of a carcinogenicity prediction program using Omics technology in human risk assessment and safety assessment during new drug development is very important in evaluating the carcinogenicity of potentially harmful substances.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to discover biomarkers specifically regulated by several classes of carcinogens, such as genotoxic / carcinogens and non-genetic / carcinogens. As a result, the present invention was completed by confirming that MOESIN has an increased expression level due to genotoxic / carcinogen treatment and has an index efficiency for genotoxic / carcinogen detection.

Accordingly, it is an object of the present invention to provide a method for detecting a carcinogen.

Another object of the present invention to provide a kit for detecting a carcinogen.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention provides a method for detecting a carcinogen comprising the following steps:

(a) contacting a test substance with a cell comprising a membrane-organizing extension spike protein (MOESIN) gene; And

(b) measuring the expression level of MOESIN protein in the cell; When the expression of the protein increases, it is determined to be a carcinogen.

According to another aspect of the present invention, the present invention provides a kit for detecting a carcinogen comprising a primer or a probe specifically binding to a membrane-organizing extension spike protein (MOESIN) gene.

According to another aspect of the present invention, the present invention provides a kit for detecting a carcinogen comprising an antibody that specifically binds to a membrane-organizing extension spike protein (MOESIN) protein.

The present inventors have made extensive efforts to discover biomarkers specifically regulated by several classes of carcinogens, such as genotoxic / carcinogens and non-genetic / carcinogens. As a result, it was confirmed that MOESIN increased the expression level by genotoxicity / carcinogen treatment, so that there was an index efficiency for genotoxicity / carcinogen detection.

MOESIN belongs to the family of ezrin-radixin-moesin (ERM) proteins. The ERM protein family acts as a link between the membrane and the cytoskeleton in actin-rich membrane structures. MOESIN is expressed in 31% of all cancers, in particular in 82% of basal cell tumors, and MOESIN expression is known to be partially involved in thyroid tumors and oral squamous cell carcinoma. In addition, MOESIN expression is a basic indicator of breast cancer, and DNA microarray methods have been used to determine that Caveolin 1, CALLA / CD10, CD44, CK5 / 6 and EGFR correlate with MOESIN expression. The above is described by Charafe-Jauffret et al ,. Int . J. Cancer . 121, 1779, 2007. Therefore, it can be confirmed that MOESIN is attracting attention as a number of tumor indicators such as breast cancer.

According to the present invention, the test substance is contacted with a cell containing the MOESIN gene.

Generally, mammalian cells contain the MOESIN gene. Cells that can be used in the present invention are primary cultured mammalian cells, established mammalian cells or cancer cell lines. Mammalian cells that can be used are, for example, mouse, rat, goat, sheep, cow, horse, pig or human cells.

In addition, cells that can be used in the present invention include cells transformed with the MOESIN gene.

The test substance, which is determined by the present invention as a carcinogen, is not particularly limited and includes a single compound or a mixture of compounds (eg, a natural extract or a cell or tissue culture). Test substances can be obtained from libraries of synthetic or natural compounds. Methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) And MycoSearch (USA).

Test materials can be obtained by various combination library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution (e.g., by a synthetic library method requiring deconvolution, a “1-bead 1-compound” library method, and a synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc . Natl . Acad . Sci . USA 90, 6909, 1993; Erb et al. Proc . Natl. Acad . Sci . USA 91, 11422, 1994; Zuckermann et al., J. Med . Chem . 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew . Chem . Int. Ed . Engl . 33, 2059, 1994; Carell et al., Angew . Chem . Int . Ed . Engl . 33, 2061; Gallop et al., J. Med . Chem . 37, 1233, 1994 and the like.

After the test substance is contacted with the cell, the expression level of the MOESIN gene (NC_000086.6) is measured in the cell. If it is determined that the expression of the gene in the cell is increased, it is determined to be a carcinogen.

“The expression level of the MOESIN gene has been increased” means that the expression level is increased compared to the expression level of the gene in untreated cells. The extent of this expression is determined by Western blotting (Sambrook, J. et al., Molecular) commonly practiced in the art. Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)) or reverse transcription-polymerase chain reaction (RT-PCR), Sambrook, J. et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold spring harbor press (2001)).

More specifically, when the expression level of 1.5 times or more is expressed compared to the expression level of the gene in untreated cells, it is determined as a significant increase in expression and the test substance is determined to be a carcinogen.

The expression level of the MOESIN gene can be measured by genetic assay or immunoassay according to a conventional procedure in the art.

When practicing the present invention based on genetic analysis, primers or probes that specifically bind to the MOESIN gene sequence are used.

In the case of using a primer, a gene amplification reaction is performed to check the expression level of the MOESIN gene. Since the present invention analyzes the expression level of the MOESIN gene, the expression level of the gene is determined by examining the mRNA amount of the MOESIN gene in the cell.

Therefore, in principle, the present invention performs a gene amplification reaction using primers that bind to mRNA or cDNA as a template of mRNA in a sample.

To obtain mRNA, total RNA is isolated from the sample. Isolation of total RNA can be carried out according to conventional methods known in the art. See Sambrook, J. et al., Molecular . Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C. et al., Plant Mol . Biol . Rep . 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology , John Willey & Sons (1987); And Chomczynski, P. et al., Anal . Biochem . 162: 156 (1987). For example, Trizol can be used to easily isolate total RNA in cells.

Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified. Since the total RNA of the present invention is isolated from human samples, it has a poly-A tail at the end of the mRNA, and cDNA can be easily synthesized using oligo dT primers and reverse transcriptases using these sequence characteristics. reference: PNAS USA, 85: 8998 ( 1988); Libert F, et al, Science, 244:... 569 (1989); and Sambrook, J. et al, Molecular Cloning A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001). Next, the synthesized cDNA is amplified through gene amplification reaction.

Primers used in the present invention are hybridized or annealed to one site of the template to form a double chain structure. Conditions for nucleic acid hybridization suitable for forming such double chain structures are described in Joseph Sambrook, et al., Molecular Cloning , A Laboratory. Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985).

Various DNA polymerases can be used for amplification of the present invention and include “Clenow” fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtained from various bacterial species, which is Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus Contains furiosus (Pfu).

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. So that the degree of amplification desired the joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{2 +} can be achieved to provide the reaction mixture is necessary.

All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process can be carried out in a single reactant without changing conditions such as addition of reactants.

Annealing or hybridization in the present invention is carried out under stringent conditions allowing specific binding between the target nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary depending on the surrounding environmental variables.

As used herein, the term "amplification reaction" means a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which are called polymerase chain reaction (hereinafter referred to as PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (hereinafter referred to as RT-PCR). (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction (LCR), Gap-LCR (WO 90 / 01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (WO 88/10315), self sustained sequence replication (WO 90/06995) ), Selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (US Pat. No. 4,437,975) ), Arbitrarily primed polymerase chain reaction (AP-PCR) (US Pat. Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA) (US Pat. 5,130,238, 5,409,818, 5,5 54,517, and 6,063,603), strand displacement amplification and loop-mediated isothermal amplification (LAMP). Other amplification methods that can be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and US Pat. No. 09 / 854,317.

In the most preferred embodiment of the invention, the amplification process is carried out according to the polymerase chain reaction (PCR) disclosed in US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159.

PCR is the most well-known method of nucleic acid amplification, and many methods have been developed that variously modify or apply this method. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase) chain reaction (IPCR), vectorette PCR, thermal asymmetric interlaced PCR (TAIL-PCR) and multiplex PCR have been developed for specific applications. For more information on PCR, see McPherson, MJ, and Moller, SG PCR . BIOS Scientific Publishers, Springer-Verlag New York Berlin, Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

The primer used in the amplification reaction in the present invention is an oligonucleotide having a sequence complementary to the cDNA sequence of the MOESIN gene. As used herein, the term “primer” refers to a single-strand that can act as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) at a suitable temperature. Oligonucleotides. Suitable lengths of primers are typically 15-30 nucleotides, although varying depending on various factors, such as temperature and the use of the primer. Short primer molecules generally require lower temperatures to form a hybrid complex that is sufficiently stable with the template.

The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer set in the present invention does not need to have a sequence that is perfectly complementary to the MOESIN cDNA sequence as a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to this sequence to act as a primer. Preferably, the primer used in the present invention has a sequence that is perfectly complementary to the MOESIN cDNA sequence.

The design of such primers can be easily carried out by those skilled in the art with reference to the cDNA sequence of the MOESIN gene, for example, using a primer design program (eg, PRIMER 3 program).

The amplified MOESIN cDNA is analyzed by a suitable method to investigate the expression level of the gene. For example, the result of the amplification reaction described above is subjected to gel electrophoresis, and the expression level of the MOESIN gene is examined by observing and analyzing the resulting band.

Through this amplification reaction, when the expression of the MOESIN gene in the cell is higher than the untreated cell, the test substance is determined to be a carcinogen.

Liver cancer can be diagnosed by hybridization-based analysis using a probe hybridized to the MOESIN gene or cDNA. As used herein, the term “probe” is a single chain nucleic acid molecule and includes a sequence that is complementary to a target nucleic acid sequence. According to one embodiment of the present invention, the probe of the present invention can be modified within a range that does not impair the advantages of the probe of the present invention, that is, the improvement of hybridization specificity. These modified labels can provide a signal that allows detection of hybridization, which can be linked to oligonucleotides. Suitable labels include fluorophores (eg fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), chromophores, chemilumines, magnetic particles, radioisotopes Elements (P ³² and S ³⁵ ), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptavidin Hapten with specific binding partners, such as, but not limited to, biotin, digoxigenin and chelating groups. Labeling can be performed in a variety of ways conventionally practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham" (1989)), and chination methods (Maxam & Gilbert, Methods). in Enzymology , 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, colorimetry, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.

According to one embodiment of the present invention, the probe hybridized to the cDNA of MOESIN may be immobilized on an insoluble carrier (eg, nitrocellulose or nylon filter, glass plate, silicon and fluorocarbon support) to prepare a microarray. In microarrays the probe is used as a hybridizable array element. Immobilization to an insoluble carrier is carried out by chemical bonding methods or by covalent binding methods such as UV. According to one specific embodiment of the present invention, the hybridization array element may be bonded to the glass surface modified to include an epoxy compound or an aldehyde group, and may also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element may be coupled to the carrier via a linker (eg, ethylene glycol oligomer and diamine).

The cDNA of MOESIN can be obtained by the above-described process. Hybridization reaction-based assays can also be performed by labeling the cDNA of MOESIN instead of the probe.

If a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization , Springer-Verlag New York Inc. NY (1999). For example, high stringency conditions were hybridized at 65 ° C in 0.5 M NaHPO ₄ , 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA, followed by addition of 0.1 x SSC / 0.1% SDS Lt; RTI ID = 0.0 > 68 C < / RTI > Alternatively, high stringency conditions means washing at < RTI ID = 0.0 > 48 C < / RTI > in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing in 0.2 x SSC / 0.1% SDS at 42 ° C.

After the hybridization reaction, the hybridization signal coming out of the hybridization reaction is detected. The hybridization signal can be performed by various methods, for example, depending on the type of label bound to the probe. For example, if the probe is labeled by an enzyme, the substrate of the enzyme can be reacted with the hybridization product to confirm hybridization. Combinations of enzymes / substrates that can be used include peroxidase (eg horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium). Nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2 , 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol / pyronine; Alkaline phosphatase with bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; Glucose oxidase, t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by silver dyeing using silver nitrate.

By analyzing the intensity of the hybridization signal by the above-described hybridization process, it is possible to determine whether the test substance is a carcinogen. In other words, when the signal treated with MOESIN cDNA is stronger in the cells treated with the test substance than in the untreated cells, the test substance is determined to be a carcinogen.

The present invention can be used to detect carcinogens by detecting MOESIN expression products, ie proteins according to immunoassay methods.

Such immunoassays can be performed according to various immunoassays or immunostaining protocols developed in the prior art. The immunoassay or immunostaining format may include Western blotting, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry , Immunofluorescence staining and immunoaffinity tablets. The immunoassay or method of immunostaining is described in Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme - linked immunosorbent assay ( ELISA ), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the method of the present invention is carried out according to a radioimmunoassay, radioisotopes (eg, C ¹⁴ , I ¹²⁵ , P ³²⁾ And antibodies labeled S ³⁵ ) can be used to detect MOESIN protein.

When the method of the invention is carried out in an ELISA manner, certain embodiments of the invention comprise the steps of: (i) coating a cell sample lysate on the surface of a solid substrate; (Ii) reacting said cell lysate with an antibody against MOESIN protein as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iii) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

Enzymes bound to the secondary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescence, luminescence or infrared reaction, for example, alkaline phosphatase, β-galactosidase, hose Radish peroxidase, luciferase and cytochrome P ₄₅₀ . When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) if colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS substrates such as (phenzaine methosulfate) may be used. All.

When the method of the invention is carried out in a capture-ELISA mode, certain embodiments of the invention comprise the steps of: (i) coating the surface of a solid substrate with an antibody against the MOESIN protein as a capturing antibody; (Ii) reacting the capture antibody with the cell sample; (Iii) reacting the product of step (ii) with a detecting antibody having a label that generates a signal and which specifically reacts with the MOESIN protein; And (iv) measuring a signal originating from said label.

The detection antibody carries a label which generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P ₄₅₀ ), radioactive substances (eg C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are available in Ed Harlow and David Lane, Using Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

When the present invention is carried out by Western blotting, a conventionally practiced method can be used (see Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 108-121, CRC press). An exemplary method of western blotting includes (i) lysing a cell sample treated with the test substance; (Ii) obtaining a protein from the lysed cells; (Iii) denaturing the obtained protein with a solution comprising SDS and 2-mercaptoethanol; (Iii) SDS-PAGE the denatured protein; (Iii) transferring the denatured protein on the gel complete with SDS-PAGE to the NC membrane; (Iii) reacting the protein on the NC membrane with an antibody against the MOESIN protein, which is the primary antibody; (Iii) reacting the secondary antibody with the primary antibody to which the enzyme catalyzing the color reaction is bound; (Iii) inducing a color reaction by adding a substrate of an enzyme bound to the secondary antibody; And (iii) measuring the degree of color development developed by the color reaction.

After Western blotting, the NC membrane with the colored bands is read directly into a densitometor, or when using a luminescent substrate (e.g. luminol), developed into a film and the film is transferred to the densitometer. The difference is quantified to determine whether the test substance is a carcinogen.

Measurement of the final enzyme activity or signal in the immunoassay method can be carried out according to various methods known in the art. Detection of these signals allows for qualitative or quantitative analysis of MOESIN proteins. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

Antibodies to the MOESIN protein used in the present invention are monoclonal antibodies.

Antibodies to the MOESIN protein can be prepared by methods commonly practiced in the art, such as by fusion methods (Kohler and Milstein, European). Journal of Immunology , 6: 511-519 (1976), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J . Mol Biol, 222:.. 58, can be prepared by 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies : A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, which is incorporated herein by reference in its entirety. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting MOESIN protein antigen into a suitable animal, collecting antiserum from the animal, and then isolating the antibody from the antiserum using known affinity techniques.

By analyzing the intensity of the final signal by the above-described immunoassay process, carcinogens can be detected. That is, when the signal for the MOESIN protein is stronger in the cells treated with the test substance than in the untreated cells, the test substance is determined to be a carcinogen.

If the kit for detecting a carcinogen of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents for RT-PCR amplification, such as buffers, reverse transcriptases, DNA polymerases, DNA polymerase cofactors and dNTPs. When the human kit of the invention is subjected to an immunoassay, the kit of the invention may optionally comprise a secondary antibody and a substrate of a label.

Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

As demonstrated in the examples below, MOESIN exhibits very good surface efficiency for carcinogens, especially for carcinogens with genotoxicity.

According to a preferred embodiment of the present invention, the carcinogen which can be identified by the present invention is a carcinogen having genotoxicity.

According to a preferred embodiment of the present invention, the carcinogen identified by the present invention is a non-genotoxic carcinogen, and step (b) is performed by measuring the expression level of the MOESIN gene. As described in the Examples below, among the markers discovered by the present invention, MOESIN shows the best index efficiency for genotoxic / carcinogen.

According to a preferred embodiment of the invention, the invention is carried out by measuring the expression of the MOESIN marker. By using such complex biomarkers, the determination of carcinogens can be made more error-free.

The features and advantages of the present invention are summarized as follows:

(a) MOESIN biomarker discovered in the present invention shows very good surface efficiency for carcinogens.

(b) The invention can be used to determine the carcinogenic potential of certain substances with improved accuracy.

(c) The present invention can also be used for safety evaluation during drug development.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experiment method

Test substance and treatment conditions

L5178Y mouse lymphoma cells (Korea Cell Line Bank) were passaged once every 2-3 days in a 37 ° C. incubator fed with 5% CO ₂ in RPMI 1640 (Gibco BRL) medium containing 10% horse serum. The treatment conditions for the test material for 2-DE (2-Dimensional Electrophoresis) are sowing the culture cells to 1 x 10 ⁶ cells / ml in the flask, and after 2 hours of treatment with the concentration of each test material, the medium was changed Cells were harvested after 22 hours of incubation. The types and concentrations of the test substances are listed in Table 1 below.

2-DE (2- Dimensional Electrophoresis )

40 μl of the protease inhibitor was added per 1 ml of the sample buffer. Samples were prepared for cells to be about 0.5-1 mg protein, and the sample buffer was added in an amount of about 1-1.5 times the sample weight. When the sample was melted, the sonication for 5 seconds and the break for 5 seconds were repeated about 15 times, and 10 μl of DNase was added to the sample and left at 4 ° C. for 30 minutes. The sample was transferred to a tube, centrifuged at 14000 rpm for 20 minutes, and then the supernatant was placed in a new tube. Proteins were quantified by spectrophotometer. For pH 3-10 (NL) 1 mg of protein was placed in a new tube and the remainder was filled with sample buffer so that the total volume was 350-400 μl.

2% of the total volume of Multiphor (GE Healthcare), an electrophoretic system, was added to an IPG (Immobilized pH gradient) buffer, left at room temperature for 30 minutes, and centrifuged at 12000 rpm for 10 minutes. Rehydration was as follows. Place the sample in a level tray groove, evenly wet the pH 3-10 (even for other pH areas) drying strips, and pour cover fluid (mineral oil) over the sample to prevent it from drying and leave for 16-20 hours. It was. Each sample was transferred to a vinyl tray groove, and a 2.5 cm electrode strip (salt leg) was placed on both ends, and the set multiphor was run for 31 hours (maintained at 20 ° C). Subsequently, the sample was transferred to a glass tube so that the gel did not touch the wall, and 10 ml of TBP equilibrium was added to each sample, and the samples were all soaked and shaken for 25 minutes at 90-95 rpm (reduction and alkylation simultaneously). How to). The sample was placed in a gel plate set and poured onto the agarose embedding solution to harden. The gel plate was fitted and 1 × SDS buffer was poured into the upper buffer by 500 ml each and 2DE was lowered. Then, the gel was removed, a fixed solution was added thereto, shaken at 40 rpm, followed by Coomassie staining for at least 12 hours, followed by destaining with distilled water.

Agarose Embedding Solution ingredient content 0.5% agarose 0.5 g 0.001% Bromophenol Blue 1 ml of 0.1% solution 1 X Running Buffer Up to 100 ml

Sample buffer ingredient content 7M Urea 21 g dry urea 2M Thiourea 7.6 g 100 mM DTE 0.77125 g 4.55% CHAPS 2 g 0.5% Carrier Ampolight 25ml or 62.5ml 40mM Tris 242mg 0.002% Bromophenol Blue Die 10 ml of 1% solution water Up to 50 ml Dispense in 1 ml tubes

TBP equilibrium ingredient content Urea 36 g SDS 2 g 5 x Tris / HCl Gel Buffer 20 ml 50% glycerol 40ml 25% acrylamide liquid 10 ml gun 100 ml

Image analysis

Coomassie stained gels were scanned on a GS-710 imaging densiometer (Bio-Rad), converted to files, and image analysis was performed using the Image Master Platinum 5 (GE Healthcare, USA) program. Protein spots were detected and volume and% vol were determined to pair each gel treated with the test substance, followed by data analysis.

Volume is a value considering the area and the peak height, and the volume for the portion corresponding to 75% of the volume, calculated from the peak, is defined as the volume to correct the possibility of inaccuracy of the spot boundary set by the spot detection.

% vol represents the percentage conversion of the volume value of a particular spot to the sum of the volume values of all the spots shown on the particular gel.

In-gel cutting

After spots were cut from the gel, the gel slices were transferred to a tube and washed with water. Subsequently, 50 μl of 50 mM NH ₄ HCO ₃ (pH 7.8): acetonitrile = 6: 4 was added to each tube and shaken, then shaken with changing the solution until the dye was completely removed, and dried in vacuo. Then, trypsin (12.5 μg / μl in 50 mM NH ₄ HCO ₃ ) was added to each tube in 10 μl and left on ice for 45 minutes, then the trypsin solution was removed and 10 μl of 50 mM NH ₄ HCO ₃ was added and reacted at 37 degreeC for 12 hours.

Desalination and Concentration of Cleaved Peptides

The flattened point was pressed about 3 mm from the end of the gel loading tip, then the tip was held and twisted once. Poros R2 resin (in 70% acetonitrile, Applied Biosystems) was placed from the top into the tip and packed to the loading tip using a 1 ml syringe (about 2-3 mm). After the packing was finished, 20 μl of 2% formic acid was added, and the resin was equilibrated by flowing in the same manner using a 1 ml syringe. 30 μl of 2% formic acid was added to 10 μl of the peptide solution and flowed to bind the resin. Subsequently, after washing with 20 μl of 2% formic acid, the peptide was spotted while eluting directly to the MALDI plate with a 0.5 μl matrix (α-cyano cinnamic acid, recrystallized) in 70% acetonitrile solution. The spotted column was washed with 100% acetonitrile and 2% formic acid.

MALDI - TOF  And Peptide Mass Fingerprinting

Peptide mass fingerprinting (PMF) was analyzed using a 4800 MALDI-TOF / TOF Mass Spectrometer (Applied Biosystems). Mass spectra were obtained in reflection / delayed extraction mode. Monoisotopic peptide mass was obtained using Data Explorer 3.5 (PerSeptive Biosystems). Proteins were identified using the MASCOT (www.matrixscience.com) search engine and were considered 95% significant when the MASCOT score was 75 or higher.

Western blotting

1. SDS-PAGE

Buffer Buffer Furtherance Nautical Gel Buffer 1.5M Tris-Cl, pH8.8 Stacking Gel Buffer 0.5M Tric-Cl, pH6.8 6X Sample Buffer 0.35M Tris-Cl, pH6.8, 10% SDS, 30% glycerol, 9.3% DTT 10X running buffer 0.025M Tris-Cl, pH8.3, 0.192M Glycine, 0.1% SDS

Cells were disrupted with sample buffer and heated at 80 ° C. or higher for 5 minutes. After the gel was fixed and running buffer was added, the running buffer was checked for leakage. The sample was then loaded into the gel, the stacking gel lowered at 80V and the resolution gel lowered to 120V. The gel was removed from the plate, the stacking portion was removed, and the buffer required for transfer was prepared.

Buffer Buffer Furtherance 10X transfer buffer Tris, glycine Transfer buffer 10X Transfer Buffer, 20% MeOH 10X TBS .0.5M Tris base, 9% NaCl, pH7.6 1X TBS 10X TBS 1% TBSt 10X TBS, Tween 20 Blocking buffer 3% Scheme Milk, 1% TBSt

The PVDF membrane and gel were soaked in the transfer buffer for 20 minutes and then fixed in the transfer mold. Then, the reaction was carried out at 240 mA for 80 minutes. The membrane was cut to size and placed in blocking buffer and allowed to react at room temperature for 1 hour. The primary antibody (Epitomics Inc.) was added to fresh blocking buffer and reacted at 4 ° C. overnight, and then washed three times with 1% TBST for 5 minutes. Secondary antibody (GE Inc.) was added to fresh blocking buffer and allowed to react at room temperature for 1 hour, followed by washing 3 times with 1% TBST. Subsequently, the substrate (enhancer solution: peroxide solution = 1: 1) was sprayed onto the film to react, and film was photographed in the dark room.

Experiment result

Genotoxic carcinogen (1,2-dibromomethane / glycydol / diethyl stilsterol / urethane / chlorambucil / dibenz anthracene / methyl methanesulfonate / nitroso methylurea / nitrosodimethylamine Nine species were treated with test substances to perform 2-DE on proteins recovered from L5178Y cells (FIG. 1).

In order to select biomarker protein candidates for genotoxicity and carcinogenicity of each class of test substance, spots of gels of each test substance were normalized and paired by image analysis to identify spot IDs for the same protein of each test substance. Relative expression change was compared with% vol (normalized vol., Corrected spot intensity) values (FIG. 2).

% Vol. Of the same Spot ID corresponding to two test substances of each class. The mean value was determined to select spots that showed an increase or decrease pattern in expression level over 2 times compared to other classes.

In the table above, G / C refers to genotoxic / carcinogenic substances.

Spot ID 397 was identified as a membrane-organizing extension spike protein (MOESIN) as a result of peptide mass fingerprinting (PMF) analysis using a MALDI-TOF / TOF mass spectrophotometer (FIG. 3). Moesin has shown an increasing pattern in genotoxic / carcinogenic substances.

Since it is easier to detect the increase pattern than the decrease pattern in the usability as a biomarker, the verification was performed on the MOESIN showing the increase pattern.

Western blotting was performed to verify the expression change of the biomarker candidate protein selected as above. Treatment conditions were treated with L5178Y cells for 2 hours in the same manner as 2-DE conditions (Fig. 4, Fig. 5).

To confirm the above results in more detail, genotoxic carcinogens (1,2-dibromomethane / glycydole / diethyl stilsterol / urethane / chlorambucil / dibenz anthracene / methyl methane Nine sulfonates / nitroso methylurea / nitrosodimethylamine) were tested for cell expression (L5178Y) every 2, 12 and 24 hours, and the efficiency of the selected indicator protein was confirmed. (FIG. 6). Based on these results, the surface efficiency was verified as shown in the table below.

In the table above, surface efficiency (%) was calculated as follows:

Pattern of increase: Number of test substances with expression growth rate of 150% or more / total number of test substances (G / C; 4, NG / C; 5) x 100

In the 2-DE results, the expression increase pattern was shown in the treatment of test substances related to genotoxicity and carcinogenicity. Measured.

Therefore, the genotoxicity of the carcinogenicity test substance may be used as a biomarker protein predicting genotoxicity / carcinogenicity-related compounds by using the expression level of MOESIN as an index. This result confirmed the reproducibility of the biomarker candidate protein that has been verified in all test substances.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a 2-DE gel photograph after 2 hours treatment of the test substance. Controls 1, 2, and 3 represent untreated, DMSO and methanol solvents, respectively, and nine genotoxic / carcinogenic test substances were 1,2-dibromomethane, glycydol, diethyl stilbestol, urethane, Chlorambucil, dibenz anthracin, methyl methanesulfonate, nitroso methylurea, nitrosodimethylamine.

FIG. 2 is a photograph obtained by analyzing the 2-DE gel of FIG. 1. In the picture, green represents dots on all gels, and red represents dots only on certain gels. The sequence of the control and genotoxic / carcinogenicity test substance is shown in FIG.

FIG. 3 is a partial 2-DE gel photograph of spot # 397 selected as a result of analyzing the image in FIG. 2. As a representative test substance, spot # 397 may be increased as compared with the untreated control when 1,2-dibromoethane and glycidol were treated.

FIG. 4 is a result of analyzing peptide mass fingerprinting (PMF) using MALDI-TOF / TOF mass spectrometer (Applied Biosystems) for spot # 397 selected as a result of analyzing the image in FIG. 2.

Figure 5 is a Western blotting picture showing the change in the expression of the protein after 2 hours of treatment. N represents no treatment, D represents DMSO, M represents a methanol solvent, and 1 to 9 are 9 genotoxic / carcinogenic test substances.

Figure 6 is a graph showing the change in protein expression of MOESIN when treated with test material for 2 hours. In the graph, the y axis represents the rate of increase and decrease of expression, and the x axis represents the test substance number. The test substance number is the same as that of FIG.

FIG. 7 is a graph of Western blotting results showing changes in protein expression during time course of treatment (2 hours, 12 hours and 24 hours). In the graph, the y axis represents the rate of change in expression, the x axis represents the treatment time of the test substance, and the test substance number is indicated on each line. The test substance number is the same as that of FIG.

Claims (7)

A method for detecting carcinogens having genotoxicity comprising the following steps: (a) contacting a test substance with a cancer cell comprising a membrane-organizing extension spike protein (MOESIN) gene; And (b) measuring the expression level of MOESIN protein in the cancer cells; Increased expression of the protein is determined to be a carcinogen with genotoxicity. delete delete The method of claim 1, wherein the expression level of the protein is measured by measuring the amount of the protein expressed from the gene. Kit for detecting carcinogens having genotoxicity, including primers or probes that specifically bind to a membrane-organizing extension spike protein (MOESIN) gene. A kit for detecting carcinogens having genotoxicity, including an antibody that specifically binds to a membrane-organizing extension spike protein (MOESIN) protein. delete

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