KR101228148B1 - Composition for diagnosis of radio-resistance or radio-sensitive marker and use thereof - Google Patents

Abstract

The present invention provides a radiation resistance or sensitivity diagnostic kit comprising a composition comprising a composition comprising a formulation for selecting a radiation resistance or sensitivity diagnostic marker and measuring the presence of the marker, and using the same to diagnose radiation resistance or sensitivity or radiation of cancer patients. The present invention relates to a method for predicting treatment.
By using the composition for diagnosing radiation resistance or sensitivity of the present invention, the radiation resistance or sensitivity of the cancer cell to be treated is determined to provide a customized radiation treatment, and the treatment effect is enhanced through the diversification of the radiation treatment target in cancer patients. Can be reduced. It can also be used to determine the prognosis (predictable therapeutic effects) of radiation therapy patients.

Description

 Composition for diagnosing radiation resistance or sensitivity comprising an agent for measuring ClCl 1 and use thereof {Composition for diagnosis of radio-resistance or radio-sensitive marker and use}

The present invention provides a radiation resistance or sensitivity diagnostic kit comprising a composition comprising a composition comprising a formulation for screening a radiation resistance or sensitivity diagnostic marker and also measuring the presence of the marker, and using the same to diagnose radiation resistance or sensitivity or radiation of cancer patients. The present invention relates to a method for predicting treatment.

Throughout the world, cancer is a serious problem that threatens human health. Many methods and techniques are currently being developed for treating or diagnosing cancer, but techniques for finding safer and more effective treatments have been developed.

Statistically, about 30-50% of cancer patients receive radiation at one time of treatment. However, the effectiveness of radiation treatments varies depending on the nature of the cancer, the patient and the radiation combined with other treatments. In general, cancers that are widely used for radiation therapy include head and neck cancer, laryngeal cancer, cervical cancer, pharyngeal cancer, lung cancer, brain cancer, breast cancer, and colon cancer. Although these carcinomas are the main targets of radiation therapy, the response of radiation therapy is often poor, so a strategy for improving the treatment efficiency is needed. In addition, although the initial response is good because the radiation treatment is good, the recurrence is frequently frequent, so it is important to improve the efficiency of radiation treatment. Although there is a difference in the sensitivity of radiation treatment to cancer cells, developing a technique that can predict radiation sensitivity for each individual before radiation treatment provides the most appropriate radiation treatment for the individual by adjusting the radiation treatment for each individual. can do.

In order to determine the sensitivity of cancer cells to radiation before radiation treatment, there is a need for a method for isolating cancer cells, irradiating them, and analyzing the extent of cancer cells accurately and quickly. The most direct and ideal method is to treat trypan blue on the cells and count the living cells using a microscope and a cell counter, but this requires time and too much effort. There is a problem. Even in patients with histological findings and stages, radiation sensitivity is very different and there are many factors that influence this. The main reason why tumors become radiation resistant is the internal radiation sensitivity of tumor cells. When cancer cells are irradiated with radiation, they die or recover from complex molecular biological changes, and mechanisms that regulate the function of genes and proteins involved in the signaling of these processes have been discovered. It is known to be a possible target (Park HJ., J Korean Soc Ther Radiol Oncol 2004; 22: S6, Choi EK., J Korean Soc Ther Radiol Oncol 2004; 22: S7). To date, studies on radiation resistance have been conducted, and some of them are based on the development of radiation sensitive agents to overcome radiation resistance. Factors related to radiation resistance that have been discovered to date include cyclooxygenase-2 (Terakado N, Shintani S, Yano J, Chunnan L, Mihara M, Nakashiro K, et al., Oral Oncol 2004; 40: 383-9), epithelium Cell Growth Factor Receptor (EGFR) (Milas L, FanZ, Andratschke NH, Ang KK., Int J Radiat Oncol Biol Phys 2004; 58 (3); 966-71), Cyclin D1 (Milas L, Akimoto T, Hunter NR , Mason KA, Buchmiller L, Yamakawa M, et al., Int J Radiat Oncol Biol Phys 2002; 52; 514-21), Insulin-Like Growth Factor 1 Receptor, Manganese Superoxide Dismutase , Survivin, etc., but the mechanism is still partially revealed, and as a useful predictor of radiotherapy results, there are few markers that can increase radiation sensitivity.

Laryngeal cancer is the sixth most common cancer in the world, the largest subgroup of head and neck cancer. Increased smoking and alcohol consumption are major risk factors for laryngeal carcinoma, and human papilloma virus is another potential factor. Radiation therapy is widely used as one of the major treatment methods to remove malignant tumors. In general, radiation therapy is combined with surgical surgery and chemotherapy to treat laryngeal cancer, and is commonly used to treat various forms of cancer. Used as Therefore, there is a need for a marker that can diagnose resistance in such laryngeal cancer.

Therefore, the present inventors have made diligent efforts to find markers for diagnosing the radiation resistance of cancer, and as a result, construct a radiation resistant cell line and analyze a radiation resistant cell line and a control cell line with a 2-D gel to identify genes having a difference in expression. Thus, the present invention has been completed with a novel radiation resistance diagnostic marker.

One object of the present invention is to provide a composition for diagnosing radiation resistance or sensitivity of cancer cells comprising an agent for measuring the level of mRNA of the CLIC1 (Chloride intracellular channel protein 1, NCBI GenBank Accession No. NM_001288) gene or a protein thereof.

Another object of the present invention is to measure mRNA levels from biological samples of cancer patients using primers that specifically bind to the CLIC1 gene; And it provides a method of providing information for diagnosing radiological resistance of cancer patients comprising comparing the change in the mRNA level with the mRNA level of the normal control sample.

Another object of the invention is to contact the antibody specific for the protein of CLIC1 with a biological sample of a cancer patient to confirm the protein level by antigen-antibody complex formation; And it provides a method of providing information for diagnosing the radiation resistance or sensitivity of cancer patients comprising the step of comparing the change in the amount of protein formation with the protein level of the normal control sample.

It is another object of the present invention to provide information necessary for predicting the prognosis of radiation therapy in cancer patients. It is to provide a method for detecting a diagnostic marker for sensitivity.

As one embodiment for achieving the above object, the present invention provides a radiation resistance or sensitivity of cancer cells comprising an agent for measuring the level of mRNA of the CLIC1 (Chloride intracellular channel protein 1, NCBI GenBank Accession No. NM_001288) gene or a protein thereof It relates to a diagnostic composition.

As used herein, the term "CLIC1 (Chloride intracellular channel protein 1)" is an intracellular chloride channel protein, which is mainly present in the nucleus of a cell and has a function of regulating cell volume, pH, cell cycle and apoptosis. . This protein has anion-selective channel activity and functions in the nucleus and plasma membrane by forming dimers or monomers. However, there is no known association between CLIC1 and radiation sensitivity and resistance. The inventors first identified the relationship between CLIC1 and radiation resistance, and according to one embodiment of the present invention, it was confirmed that CLIC1 expression was reduced in radiation resistant cell lines, and that CLIC1 was a radiation sensitive gene.

In addition, the radiation-resistant or radiation-sensitive diagnostic marker is APRT1 (Adenine phosphoribosyltransferase, NCBI GenBank Accession No. NM_000485), PRDX2 (Peroxiredoxin-II, NCBI GenBank Accession No. NM_005809), TRM112L (TRM112-like protein, NCBI GenBank Accession No.NM_016404), PCBP2 (Poly (rC) -binding protein 2, NCBI GenBank Accession No. NM_001098620), TALDO1 (Transaldolase-1, NCBI GenBank Accession No. NM_006755), ECH1 (Enoyl Coenzyme A hydratase 1, NCBI GenBank Accession No. NM_001398), PSMA1 (Proteasome subunit alpha type-1, NCBI GenBank Accession No. NM_148976), PRPS2 (Phosphoribosyl pyrophosphate synthetase 2, NCBI GenBank Accession No. NM_001039091), NP (Nucleoside phosphorylase, NCBI GenBank Accession No. 270 No.). PSMA6 (Proteasome subunit alpha type-6, NCBI GenBank Accession No. NM_002791), TPI1 (Triosephosphate isomerase, NCBI GenBank Accession No. NM_000365), ALD (Aldehyde reductase, NCBI GenBank Accession No. NM_006066), EB1 (End-binding protein 1) , NCBI GenBank Acc ession No. NM_012325), TCP1 (T-complex 1, NCBI GenBank Accession No. NM_030752) and PDIA3 (Protein disulfide-isomerase A3, NCBI GenBank Accession No. NM_005313) relates to a composition for diagnosing radiation resistance of cancer cells comprising an agent for measuring the level of mRNA or protein of one or more genes selected from the group consisting of desirable.

The term "radio-resistance" in the present invention refers to a state that is not easily affected by irradiation and has little change in cell repair or proliferation upon exposure to radiation.

The term "radio-sensitive" in the present invention refers to a state that is easily affected by irradiation and shows a change in cell repair or proliferation upon exposure to radiation.

The composition for diagnosing radiation resistance or sensitivity of the present invention is composed of genes having differential expression levels in radiation resistant cancer cells compared to normal cancer cells, thereby determining the radiation resistance of the individual or cells. That is, an individual or cancer cells whose expression level is higher or lower than the control group may be determined to have radiation resistance or sensitivity.

In the embodiment of the present invention, the radiation sensitive diagnostic markers are expressed in CLIC1 (Chloride intracellular channel protein 1) and EB1 (End-binding protein 1), and the expression level of the radiation sensitive diagnostic marker in a radiation resistant cancer cell line is lower than that of the control group. On the other hand, diagnostic markers for radiation resistance include APRT1 (Adenine phosphoribosyltransferase), PRDX2 (Peroxiredoxin-II), TRM112L (TRM112-like protein), PCBP2 (Poly (rC) -binding protein 2), TALDO1 (Transaldolase-t), and ECH1. (Enoyl Coenzyme A hydratase 1), PSMA1 (Proteasome subunit alpha tvpe-1) PRPS2 (Phosphoribosyl pyrophosphate synthetase 2), NP (Nucleoside phosphorylase), PSMA6 (Proteasome subunit alpha type-6), TPI1 (Triosephosphate isomerase), ALD (Aldede) The expression level of the diagnostic marker for radiation resistance in radiation-resistant cancer cell lines was higher than that of the control group with reductase, EB1 (End-binding protein 1), TCP1 (T-complex 1) or PDIA3 (Protein disulfide-isomerase A3). It was OK.

If the radiation resistance or sensitivity of the cancer to be treated is judged and the level thereof is determined, the radiation dose of the radiation treatment can be determined in accordance with the determination, so that the efficiency and treatment result of the radiation treatment can be improved.

As used herein, the term "marker or diagnostic marker" refers to a substance capable of diagnosing a cancer cell or an individual having a cancer disease from a radiation resistant cell or an individual, a radiation-sensitive cell or an individual, and is capable of diagnosing a normal cancer cell. Organic biomolecules, such as polypeptides, proteins or nucleic acids (such as mRNA), lipids, glycolipids, glycoproteins or sugars (such as monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in cancer cells or individuals that are radiation resistant in comparison. Include them.

In addition, cancers that can be measured by the composition of the present invention include cancers that can be distinguished by the expression difference of the marker gene of the present invention without limitation, preferably head and neck cancer, laryngeal cancer, cervical cancer, pharyngeal cancer, lung cancer , Brain cancer, breast cancer, colon cancer and the like, more preferably head and neck cancer or laryngeal cancer.

Gene expression levels in biological samples can be confirmed by identifying the amount of mRNA or protein.

In the present invention, the term "mRNA expression level measurement" is to measure the amount of mRNA in the process of confirming the presence and expression of mRNA of the marker genes in a biological sample to diagnose radiation resistance and sensitivity. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

In the present invention, "measurement of protein expression level" is a process of confirming the presence and expression level of a protein expressed from the marker gene in a biological sample to diagnose radiation resistance or sensitivity, and preferably, the protein of the gene The amount of protein can be determined using an antibody that binds specifically to the antibody. Analytical methods for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc., but are not limited to these. .

The agent for measuring the level of the gene mRNA of the present invention can be produced by a person skilled in the art using a variety of programs based on the sequence of the gene known in the known database to the primer specifically binding to the gene.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3 'terminal hydroxyl group, capable of forming base pairs with complementary templates and serving as a starting point for template strand copying. do. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. Primers of the invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis. The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, phosphoroamidate , Carbamate, etc.) or charged linkages such as phosphorothioate, phosphorodithioate and the like. Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents. Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.

 In addition, the agent for measuring the protein level of the gene is a composition for diagnosing radiation resistance or sensitivity of cancer cells comprising an antibody specific for the protein.

As used herein, the term "antibody" means a specific protein molecule directed to an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Since radiation resistant and sensitive marker proteins have been identified as described above, the production of antibodies using them can be readily prepared using techniques well known in the art. Polyclonal antibodies can be produced by methods well known in the art for injecting the radiation resistant or sensitive marker protein antigens described above into an animal and collecting blood from the animal to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like. Monoclonal antibodies are known in the art by the hybridoma method (see Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature, 352: 624). -628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991). Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The antibodies of the present invention also include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.

As used herein, the term "cancer cell" refers to any cell that exhibits unregulated growth in the tissues or organs of a multicellular organism.

The radiation resistance diagnostic marker of the present invention was established by establishing a radiation resistant cell line using a laryngeal cancer cell line and then comparing the amount of gene expression with a control (normal laryngeal cancer cell; HEp-2 cells) by 2D-PAGE method. It can be used to enhance the effectiveness of the treatment.

According to one embodiment of the present invention, in order to construct a radiation resistant cell line, the step of irradiating the laryngeal cancer cell line with 2 Gy gamma rays twice per week for 15 weeks was repeated until reaching 60 Gy. Radiation resistance was determined by colony formation assay for the radiation resistant cell lines established through this method. Radiation resistant cell lines and control cell lines were tested by 2D-PAGE analysis to identify genes with differences in expression between the two cell lines. Genes identified as having lower expression levels in radiation-resistant cell lines compared to controls showed nucleotide sequences as opposed to CLIC1 or EB1. As a result of analysis, all 16 were APRT1, PRDX2, TRM112L, PCBP2, TALDO1, ECH1, PSMA1, PRPS2, NP, PSMA6, TPI1, ALD, TCP1 or PDIA3. These genes have been identified as novel radiation resistance or sensitivity diagnostic markers that have not yet been described in the literature as having radiation-specific, radiation-sensitive and sensitivity-related functions.

As used herein, the term "irradiation" includes exposure to the action of electromagnetic radiation or alpha or beta particles, including, for example, X-rays, ultraviolet light, alpha particles and gamma rays.

As used herein, the term "Gy" is the amount of radiation that releases 1 J in 1 kg of representative biological tissue.

As another aspect, the present invention relates to a radiation resistant or sensitive diagnostic kit comprising the radiation resistant or sensitive diagnostic composition. The kit is characterized in that the RT-PCR kit, DNA chip kit, microarray or protein chip kit. The diagnostic kit according to the present invention may be prepared according to conventional methods used in the art using the radiation resistant or sensitive diagnostic composition. In addition, measurement of the expression change of the radiation-resistant or sensitive gene can be performed using a kit comprising a radiation-resistant or radiation-sensitive diagnostic marker according to the present invention.

As used herein, the term "kit" refers to mRNA expression levels or proteins of CLIC1, EB1, APRT1, PRDX2, TRM112L, PCBP2, TALDO1, ECH1, PSMA1, PRPS2, NP, PSMA6, TPI1, ALD, TCP1 or PDIA3 genes. By means of confirming the expression level means a tool that can diagnose whether cancer cells are radiation resistant or sensitive. In the kit for diagnosing radiation resistance of cancer cells of the present invention, as a radiation resistance diagnostic marker, CLIC1, EB1, APRT1, PRDX2, TRM112L, PCBP2, TALDO1, ECH1, PSMA1, PRPS2, NP, PSMA6, TPI1, ALD, TCP1 or PDIA3 gene expression level Primers, probes or optionally antibodies that recognize markers, as well as one or more other component compositions, solutions, or devices suitable for the assay method may be included.

In another aspect, the present invention provides a method for preparing mRNA, comprising the steps of: measuring mRNA levels from biological samples of cancer patients using primers that specifically bind to the CLIC1 gene; And it relates to a method of providing information for diagnosing radiological resistance or sensitivity of cancer patients comprising comparing the change in the mRNA level with the mRNA level of the normal control sample. And further comprising measuring and comparing mRNA levels of one or more genes selected from APRT1, PRDX2, TRM112L, PCBP2, TALDO1, ECH1, PSMA1, PRPS2, NP, PSMA6, TPI1, ALD, TCP1, and PDIA3. It is desirable to include a method of providing information for diagnosing radiation resistance or sensitivity of a patient.

As another aspect, the present invention comprises the steps of contacting an antibody specific for the protein of CLIC1 with a biological sample of a cancer patient to confirm the protein level by antigen-antibody complex formation; And it relates to a method of providing information for diagnosing radiation resistance or sensitivity of cancer patients comprising comparing the change in the amount of protein formation with the protein level of a normal control sample. And further measuring and comparing protein levels of one or more genes selected from APRT1, PRDX2, TRM112L, PCBP2, TALDO1, ECH1, PSMA1, PRPS2, NP, PSMA6, TPI1, ALD, TCP1 or PDIA3. It is desirable to include a method of providing information for diagnosing radiation resistance or sensitivity of a patient.

For the purposes of the present invention, if the expression levels of mRNA and protein of the CLIC1 and EB1 genes in cancer cells are low, they can be determined to be cancer cells that exhibit radiation resistance, whereas if the expression levels of the genes are high, they can be determined to be cancer cells that exhibit radiation sensitivity. have. In addition, high mRNA and protein expression levels of APRT1, PRDX2, TRM112L, PCBP2, TALDO1, ECH1, PSMA1, PRPS2, NP, PSMA6, TPI1, ALD, TCP1 or PDIA3 genes may be considered to be radiation-resistant cancer cells. If low, it can be considered as a radiation-sensitive cancer cell line.

In another aspect, the present invention provides a method for predicting the prognosis of cancer patients in cancer, in which the radiation level of the mRNA or protein thereof of CLIC1 is compared with the expression level of normal cells in a sample of patients receiving radiation therapy. The present invention relates to a method for detecting a resistance diagnostic marker. In addition, further measuring and comparing the mRNA or protein levels of at least one gene selected from APRT1, PRDX2, TRM112L, PCBP2, TALDO1, ECH1, PSMA1, PRPS2, NP, PSMA6, TPI1, ALD, TCP1 or PDIA3. It is preferable to include a method for detecting a radiation resistance or sensitivity diagnostic marker comprising. In the method of the present invention, the cancer cells may be derived from head and neck cancer, laryngeal cancer, cervical cancer, pharyngeal cancer, lung cancer, brain cancer, breast cancer, colon cancer, but is not limited thereto. Preferably the cancer cells are from head and neck cancer or laryngeal cancer.

As used herein, the term "prognostic prediction" refers to predicting radiation-resistant or sensitive cancer cells through genes showing expression differences in radiation-resistant cancer cell lines after irradiation.

As used herein, the term "chemotherapy inhibitor" includes any chemical used to treat or control a disease. Examples of cancer chemotherapy inhibitors include, but are not limited to, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or indanyloxyacetic acid-94 (IAA94).

As used herein, the term “differential expression” refers to the expression level of the same one or more biomarkers of the present invention in a sample, by measuring the amount or level of mRNA of the biomarker in one sample, thereby determining one or more of the present invention. The difference in the expression level of mRNA of the biomarker. “Differentially expressed” may also include the determination of a protein encoded by a biomarker of the invention in a sample or sample population as compared to the amount or level of protein expression in the sample population. Differential expression can be measured as described herein and as understood by one of ordinary skill in the art.

As used herein, the term "gene expression pattern" or "gene expression profile" or "gene characterization" refers to the relative expression of genes involved in the radiation resistance and sensitivity of cancer cells, wherein the combined pattern is 3, 4 of the biomarkers of the present invention. Analysis results of expression levels of one or more biomarkers of the invention, including 5, 6, 7, 8, 9, 10, 11, 12 or all. Gene expression patterns or gene expression profiles or gene features can be obtained from the measurement of expression of mRNA and / or proteins expressed by genes corresponding to the biomarkers of the present invention.

In the embodiment of the present invention was examined the relationship between the function of CLIC1 and role ROS generation. More specifically, Western blot and immunofluorescence optical microscopy analysis of the expression of CLIC1 in parental HEp-2 and RR-HEp-2 cells confirmed that CLIC1 is a negative regulator of radiation resistance obtained in laryngeal cancer cells. Chemical inhibitors were used to examine the role of CLIC1. Therefore, it was found that inhibition of CLIC1 activity and inhibition of expression play an important role in obtaining a radiation resistant phenotype of HEp-2 cells.

In addition, overexpression of CLIC1 overexpression showed that CLIC1 contributed to the radiation sensitivity of HEp-2 laryngeal cancer cells, and that CLIC1 was expressed in a cell line showing radiation sensitivity. In the cell lines showing low expression of CLIC1, it was found that CLIC1 expression plays an important role in radiation sensitivity and is highly related to radiosensitivity and resistance in cancer cell lines. In addition, CLIC1 acts as a positive regulator of ROS production by measuring the relative DCF values of ROS generation in each HEP-2 and RR-HEp-2 cells using DCF staining. It was found that the regulation of the delivery pathway could alter the radiation sensitivity of HEp-2 cells.

By using the composition for diagnosing radiation resistance or sensitivity of the present invention, the radiation resistance or sensitivity of the cancer cell to be treated is determined to provide a customized radiation treatment, and the treatment effect is enhanced through the diversification of the radiation treatment target in cancer patients. Can be reduced. It can also be used to determine the prognosis (predictable therapeutic effects) of radiation therapy patients.

1 relates to the establishment of a radiation resistant HEp-2 human laryngeal cancer cell line. (A) The parental HEp-2, RR-HEp-2 and RR-HEp-2 variants (# 6, 12, 13 and 18) were exposed to known radiation doses to show colony-forming survival fractions. Survival fractions were determined by the following formula: number of colonies generated after radiation treatment / number of seeded cells x calculated by the plating efficiency of the control group. (B) The parental HEp-2, RR-HEp-2 and RR- # 6 cells were visualized by trypan-blue staining after 0 or 4 Gy irradiation of the colonies. (C) Parent HEp-2, RR-HEp-2 and RR- # 6 cells were incubated for 24 hours after 0 or 10 Gy of irradiation and EGFR, ERBB-2, Hsp90, Bcl-xl and Mn-SOD Transcription levels were measured by typical RT-PCR. GAPDH was used as loading control. (D) Parent HEp-2, RR-HEp-2 and RR- # 6 cells were incubated for 24 hours after 0 or 10 Gy of irradiation and EGFR, ERBB-2, Hsp90, Bcl-xl and Mn-SOD Transcription levels were measured by quantitative RT-PCR.
2 shows the effect of radiation on cell proliferation and apoptosis in parental HEp-2, RR-HEp-2 and RR-HEp-2 variant cells. (A) Cells were plated at a density of 2 × 10 ⁴ cells on a 60 mm plate, irradiated with 0 (control) or 4 Gy, incubated on a known date, and the total number of living cells was counted with trypan blue solution. (B) The cells were irradiated with 0 or 10 Gy and cultured for 48 hours. Cell viability was then measured with a FACScan flow cytometer and expressed as percentage of PI-positive cells (apoptosis). (C) The cells were cultured for 48 hours after 0 or 10 Gy irradiation. Then, the protein level of the cleaved PARP was measured by Western blot (top) and the degree of nuclear blebbing was measured by DAPI staining (bottom). β-actin was used as loading control.
Figure 3 shows the results of two-dimensional gel analysis of proteins showing differential expression between the parent HEp-2 cell line and RR-HEp-2 variant cells. (A) The parental HEp-2 cell line and RR-HEp-2 cells were incubated for 48 hours, and lysates were collected from each cell line. 150 μg of each protein was separated on a pH 4-7 gradient strip of immobilized 12% polyacrylamide gel. Proteins were visualized by silver staining and expressed using PdQuest software. Protein spots showing differential expression patterns were marked with black arrows and numbered. (B) An enlarged view of the 16 identified spots indicated in A. 15 protein spots known in (C) A were measured by Western blot. β-actin was used as loading control. The transcription level of spot 8 (TRM112L), known in (D) A, was measured by typical RT-PCR. GADPH was used as a loading control. (E) Parent HEp-2 and RR-HEp-2 variants (# 6, 12, 13 and 18) cells were incubated for 48 hours, and cell lysates of each cell line were collected. Proteins were visualized by silver staining, and magnified images of RR-HEp-2 subtype clones isolated from HEp-2 cells were shown as relative ratios of each protein expression.
4 shows expression of CLIC1 in parental HEp-2, RR-HEp-2 and RR- # 6 cells. (A) Magnification of spot 2 known in FIG. 3A (above). This was named CLIC1 and confirmed by MALDI-TOF mass spectrometer analysis (below). (B) The parent HEp-2, RR-HEp-2 and RR- # 6 cells were incubated for 48 hours, and the expression levels of the protein (top) and mRNA (bottom) of CLIC1 are shown by Western blot and RT-PCR. β-actin was used as a loading control, GADPH was used as an intracellular control. (C) After 48 hours of incubation of parent HEp-2 and RR-HEp-2 cells, optical images of CLIC 1 expression in each cell were shown (left), and the nuclei were visualized by DAPI staining (right). ). (D) Parent HEp-2 cell line was irradiated with 10 Gy radiation and then cultured for a known time (top) or 24 hours (bottom). Protein levels and cleaved PARP levels of CLIC1 were measured by Western blot and β-actin was used as loading control. Optical images of CLIC1 were immunofluorescent stained, and the nuclei were visualized using DAPI.
Figure 5 shows blocking of apoptosis by CLIC1 inhibition in parental HEp-2 cell line after (A) irradiation. (B) CON represents the control group not irradiated, VEH represents the carrier alone irradiated with 10 Gy of radiation, after 48 hours incubation in the presence of 100 mM DIDS, 10 mM IAA94 or 5 mM NAC, Expression of cleaved PARP and CLIC1 in -2 cell line was measured by Western blot. (C) HEp-2 cells were irradiated with each known radiation dose, and survival fractions were shown by comparison between the presence and absence of 10 μM of IAA94 and CON. (D to F) HEp-2 cells were transfected with control siRNA (siCON) or 100 nM CLIC1 siRNA (siCLIC1) transfected group for 48 hours, and then irradiated with 10 Gy of radiation 48 hours were further incubated (D and E). (F) was irradiated with a known radiation dose. Cell viability was measured using a FACScan flow cytometer and expressed as percentage of PI-positive cells (A to D). Protein levels of cleaved PARP and protein expression levels of CLIC1 were measured by Western blot (B and E). β-actin was used as loading control. Colony forming survival fractions were colonized by trypan blue staining and quantified by automated colony counters (C and F).
Figure 6 shows the apoptosis induced by overexpression of CLIC1 outside the normal place in RR-HEp-2 cells after irradiation. CON transfected RR-HEp-2 cells with empty vector, CLIC1-myc transfected myc-tagged CLIC1 expression vector. The cells were incubated for 24 hours and then irradiated with 10 Gy, followed by an additional 48 hours. Cell morphology was observed using (A) light microscopy, and (B) cell viability was measured using a FACScan flow cytometer, expressed as a percentage of PI-positive cells. (C) Protein levels of cleaved PARP and protein expression levels of CLIC1 were measured by Western blot. (D) β-actin was used as loading control. Colony-forming survival fractions were colonized by trypan blue staining and quantified by automated colony counters.
Figure 7 shows the function of CLIC1 in five different squamous cell lines. (A) SW756, NCI-H596, SiHa, SNU899 and SNU1076 cells were incubated for 48 hours (above). 0 or 4 Gy of radiation was irradiated (below). Protein expression levels of CLIC1 were measured by Western blot (above). Colony formation was quantified by automated colony counters (below). β-actin was used as loading control (B and C). (B) CON is transfected SNU1076 cells with an empty vector, and CLIC1-myc is transfected with SNU1076 cells with a myc-tagged CLIC1 expression vector. The cells were incubated for 24 hours. (C) The group in which the control siRNA (siCON) was transfected into SW756H cells or the group transfected with 100 nM CLIC1 siRNA (siCLIC1) was incubated for 48 hours. They were incubated for an additional 48 hours after irradiation with 10 Gy of radiation. Cleaved PARP protein levels and Myc (CLIC1) and CLIC1 were measured via Western blot (above). β-actin was used as loading control. Cell viability was measured using a FACScan flow cytometer and expressed as percentage of PI-positive cells (below).
8 shows the association between CLIC1 and ROS generation in response to radiation in HEp-2 cells. (A) After irradiating 0 or 10 Gy of radiation in parental HEp-2, RR-HEp-2 and RR- # 6 cells, DCF staining was used 12 hours later to express the ROS production of cells as relative DCF values. . (B) ROS production of cells in the presence of VEH, 10 μM IAA94 treated with 0 Gy (CON) or 10 Gy radiation in HEp-2 cells showed relative levels of DCF staining. NAC 5 mM was used as a positive control to reduce radiation-induced ROS production. (C to E) HEp-2 cells were cultured for 48 hours in the group transfected with control siRNA (siCON) or the group transfected with 100 nM CLIC1 siRNA (siCLIC1). After irradiation with 0 or 10 Gy of radiation, DCF staining was used 12 hours later to express the ROS production of cells as relative DCF values. (D and F) CON transfected RR-HEp-2 cells with empty vector and CLIC1-myc transfected with myc-tagged CLIC1 expression vector. It was incubated for 24 hours. After irradiation with 0 or 10 Gy of radiation, DCF staining was used 12 hours later to express the ROS production of cells as relative DCF values. Transcription levels of CLIC1, Mn-SOD and PRDX2 were measured by quantitative RT-PCR and typical RT-PCR. GAPDH was used as loading control.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are presented for illustrative purposes and should not be construed as limiting the scope of the invention.

Example 1. Preparation and Treatment of Cells

Human laryngeal cancer cell lines HEp-2 cells and human cervical cancer cell lines SiHa and SW756 cells and human squamous epithelial bronchial cancer cells NCI-H596 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Human laryngeal cancer cell lines SNU-899 and SNU-1076 cells were obtained from Korea Cell Line Bank. HEp-2, SiHa and SW756 cells were cultured in DMEM (Gibco-BRL, Rockville, MD, USA) medium containing 10% FBS, and NCI-H596, SNU899 and SNU1076 cells were RPMI 1640 medium containing 10% FBS Incubated at. All cells were cultured in a 37 ° C. incubator with 5% CO ₂ . Cells were irradiated with ¹³⁷ Cs (Atomic Energy of Canada, Mississauga, Canada) at 3.81 Gy / min radiation dose.

In the inhibitor experiments, the following chemicals were treated for 30 minutes prior to irradiation. This is to inhibit the activity of CLIC1, using DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and IAA94 (indanyloxyacetic acid-94). N-acetyl cysteine (NAC) was used to see ROS production.

Example 2. Establishment and Characterization of Radioresistant Human HEp-2 Laryngeal Cancer Cell Line

To establish a radioresistant laryngeal cancer cell line, HEp-2 cells were plated in 100 mm dishes at a density of 5 × 10 ³ cells / cm ² and cultured for one day. HEp-2 cells were then exposed to 2 Gy of radiation twice a week for 15 weeks until the total radiation dose accumulated at 60 Gy. When 80-90% of the irradiated cells grew, they were subcultured, followed by irradiation.

2-1. Colony Formation Analysis

Radiation sensitivity was determined by colonogenic assay. More specifically, the parental HEp-2 cell line or the RR-HEp-2 cell line was irradiated with radiation doses ranging from 0 to 6 Gy alone. Immediately after irradiation, cells were trypsinized, detached and diluted, and then cells were plated at various densities in 60 mm tissue culture dishes. 200 cells for the control group, 400 cells for the 2 Gy radiation treated cell group, 1500 cells for the 4 Gy radiation treated cell group, and 3000 cells for the 6 Gy radiation treated cell group were laid. After 10-14 days, colonies were fixed with 99.5% methanol and stained with trypan blue solution for observation.

As a result, the irradiated HEp-2 cell population (hereinafter referred to as 'RR-HEp-2') and the subtype clones isolated from the RR-HEp-2 cell population (RR- # 6, 12, 13 and 18) were separated from the parent HEp. The radiation resistance in the range of 0-6Gy showed higher radiation resistance than -2 cells (control cells) (FIG. 1A). After 4 Gy irradiation, colonies were formed, which could be seen through trypan blue staining. In particular, a significant increase was observed in RR-HEp-2 cells and RR- # 6 cells compared to the parental HEp-2 cells (FIG. 1B).

2-2. Typical PCR and Quantitative PCR Analysis

Total RNA was isolated using RNA STAT-60 (Tel-Test B, Friendswood, TX, USA). Reverse transcription reaction was performed using ImProm-IITM reverse electronenzyme (Promega) for 5 minutes of binding reaction at 70 ° C. and first strand extension reaction at 42 ° C. for 60 minutes. For typical PCR analysis, the experiment was conducted using the following primers and reaction conditions. Primers for CLIC1 are primer pairs of sense 5'-atggctgaagaacaaccg-3 '(SEQ ID NO: 1) and antisense 5'-ttatttgagggactttga-3' (SEQ ID NO: 2); primers for Bcl-xl are sense 5'-cgggcattcagtgacctgac-3 Primer pairs of '(SEQ ID NO: 3) and antisense 5'-tcaggaaccagcggttgaag-3' (SEQ ID NO: 4), primers for EGFR are sense 5'-cgtcgctatcaaggaattaag-3 '(SEQ ID NO: 5) and antisense 5'-tggtgggtatagattctgtg-3 Primer pairs of '(SEQ ID NO: 6), primers for GAPDH are primer pairs of sense 5'-catctctgccccctctgctga-3' (SEQ ID NO: 7) and antisense 5'-ggatgaccttgcccacagcct-3 '(SEQ ID NO: 8), primers for ERBB2 Is a primer pair of sense 5'-catatgtctcccgccttctg-3 '(SEQ ID NO: 9) and antisense 5'-cccacacagtcacaccataac-3' (SEQ ID NO: 10), primers for Hsp90 are sense 5'-ttggaggaacgaagaataaagg-3 '(SEQ ID NO: 11) And a primer pair of antisense 5'-gggctctgaattccaactgtc-3 '(SEQ ID NO: 12), Mn-SOD Primers for the sense 5'-tgttacagcccagatagc-3 '(SEQ ID NO: 13) and antisense 5'-cagttacattctcccagttg-3' (SEQ ID NO: 14), primers for PRDX2 are the sense 5'-aaagggaagtacgtggtc-3 '(SEQ ID NO: 15) and a primer pair of antisense 5'-gggcttaatcgtgtcactg-3 '(SEQ ID NO: 16), primers for the TRM112-like protein are sense 5'-cttacccacaatctgctg-3' (SEQ ID NO: 17) and antisense 5'-gaacatacgtccagattcc-3 ' The primer pair of (SEQ ID NO: 18) was used to perform a binding reaction at 55 ° C. and reacted 31 times.

For quantitative real-time PCR, experiments were performed using chromo 4 cycler (Bio-Rad) and SYBR Premix ExTaqTM (Takara Bio, Shiga, Japan), and the amplified signal normalized the target gene for GAPDH.

In order to confirm the radiation resistance phenotype of RR-HEp-2 cells, we compared the transcription patterns of several regulatory signal molecules of EGFR, Bcl-2, Hsp and SOD systems related to cancer radiation resistance in radiotherapy (FIG. 1C). Typical PCR results showed a significant increase in RR-HEp-2 cells and RR- # 6 cells compared to the parental HEp-2 cells, consistent with quantitative real-time PCR data (FIG. 1D). .

2-3. Cell Proliferation Analysis

To investigate the effect of irradiation on cell proliferation and death of parental HEp-2 and RR-HEp-2 cells, unradiated cells and 4 or 10 Gy-irradiated cells were cultured for a specified period of time as a control. . Compared to the parental HEp-2 cells in both the control group and the irradiated group, it was found that the cells proliferated more actively in the RR-HEp-2 cells and the RR- # 6 cells (FIG. 2A). This suggests that the established RR-HEp-2 cell line has the ability to overcome the growth inhibitory effects of radiation.

2-4. Apoptosis Analysis with FACS

Irradiation of 10 Gy caused apoptosis of the parental HEp-2 cell line. On the other hand, in the RR-HEp-2 cell line including the RR- # 6 and RR- # 13 subgroups, radiation mediated apoptosis was suppressed through FACS analysis (FIG. 2B).

2-5. Apoptosis Analysis by Western Blot

The cells were lysed in a buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40 and 0.1% SDS. Proteins were separated on SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked with 5% skim milk powder, and the secondary antibody was attached at room temperature for 1 hour. Then, the following antibodies were used and used for detection. Rabbit polyclonal anti-cut poly ADP-ribose polymerase (PARP) (Asp214) (Cell Signaling Technology, Beverly, MA, USA), mouse monoclonal anti-betaactin (Sigma), mouse monoclonal anti- CLIC1, anti-EB1, anti-PSMA1, anti-PSMA6, anti-TPI1, anti-AKR1A1 (ALD) and anti-PDIA3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-ECH1 (Santa Cruz Biotechnology), Goat Polyclonal Anti-NP and Anti-TCP1 (Santa Cruz Biotechnology), Mouse Polyclonal Anti-APRT and Anti-TALDO1 (Abcam, Cambridge, UK), Rabbit Polyclonal Anti-PRPS2 (Abcam) , Rabbit polyclonal anti-PRDX2 (Lab frontier, Seoul, Korea), and mouse monoclonal anti-PCBP2 (ABGENT, San Diego, CA, USA). Blots were developed using secondary antibodies conjugated with peroxidase and an ECL system (Amersham Life Sciences, Piscataway, NJ, USA). Radiation mediated apoptosis was inhibited in the RR-HEp-2 cell line even with the level of cleaved PARP, a typical marker of apoptosis, and PI (propidium iodide) staining (FIG. 2C) of nuclear blebbing. . These results confirm that the established RR-HEp2 cell line has a radioresistant phenotype, confirming typical physiological and molecular characteristics.

Example 3 Identification of Differential Protein Expression Patterns in Parent HEp-2 Cell Line and RR-HEp-2 Cells

3-1. 2-D PAGE analysis

In order to identify proteins whose expression is regulated differently in RR-HEp cells or isolated subtype clones, protein expression profiles of the parental HEp-2 cell line and all RR-HEp-2 cell populations were confirmed by 2-D PAGE analysis.

After dissolving the parental HEp-2 cell line and the RR-HEp-2 cell line in 200 µl of 2-D PAGE buffer, the cells were crushed by ultrasonication using a probe ultrasonic cell crusher (Branson Ultrasonic Corporation, Danbury, USA). Centrifugation for 15 minutes at 12000 rpm. Protein concentrations were measured by Micro Bradford Assay (Bio-Rad Laboratories, Hercules, Calif., USA) and each 150 μg of cell lysate was mixed with rehydration buffer. The first one-dimensional separation was performed using the IPGphor isoelectric point system in the IPG strip. The total focusing time is 60000Vh, and after focusing, the strip is in equilibrium. Two-dimensional electrophoresis was performed at 50 mA for 5 hours using 12.5% SDS-PAGE (18.3 × 20 cm) in a PROTEAN II xi 2-D cell (Bio-Rad Laboratories) system. All protein spots of the 2-D gel were visualized using silver-staining method after SDS-PAGE. More specifically, after 2-D PAGE, the gel was fixed in the first solution containing 40% ethanol and 10% acetic acid for 30 minutes. The fixed gel was treated for 30 min in a second solution containing 30% ethanol and 0.1% glutaaldehyde, 0.2% sodium thiosulfate and 6.8% sodium acetate (w / w). The gel was then washed three times for 5 minutes in secondary distilled water. The washed gel was reacted with a third solution containing 0.25% silver nitrate and 0.01% formaldehyde. The gel was then washed for 2 minutes in secondary distilled water. Spots were visualized with a solution containing 2.5% sodium carbonate and 0.01% formaldehyde. The development was stopped with 1.5% EDTA-2Na2H ₂ O. After silver staining, the gel was scanned at 200 resolution and analyzed by Imagemaster ^™ 2D platinum software (Amersham Bioscience).

As a result, an average of 850 proteins could be observed in each gel by Imagemaster ^™ 2D platinum software, and four independent experiments were performed to select 20 protein spots with a change of 1.5 times or more (FIG. 3A). 20 proteins that differ from the parent cell line are shown in Table 1.

3-2. MALDI - TOF  analysis

Of the 20 proteins, 16 proteins were successfully identified by MALDI-TOF mass MS analysis.

The MALDI-TOF mass MS analysis method is as follows. First, differentially expressed protein spots were cut out of the gel. All spots were desalted silver and the gels were digested with trypsin. Dry trypsin-treated peptides on MALDI-TOF plates were simultaneously applied to a matrix composed of CHCA (Sigma-Aldrich Chemie, Taufkirchen, Germany) solution. The MS was analyzed using a Voyager-DETM STR Workstation (Applied Biosystems, Framingham, Mass., USA) in MS mode. Each spectrum was opened in DataExplorer ^™ (version 4.5, Applied Biosystems) software, and those with the highest mass below 60 were selected by S / N threshold initial setting and modified with improved baseline. Spectra were measured with two or more peptides due to trypsin autolysis (m / z 842.5100, 2211.1046 and 2283.1807). The peak list is copied to the Mascot public interface, and very intense trypsin peaks (m / z 842.5100, 1045.5642, 2211.1046 and 2283.1807) are excluded from the peak list. Protein identification was obtained by PMF based on metric data with serial mass spectra, and information was obtained from the Swiss-Prot protein database. The edited peak list was retrieved by the Swiss-Prot database.

As a result, each gel image of HEp-2 cells and RR-HEp-2 cells was enlarged and shown (FIG. 3B). Of the 16 proteins identified, 14 proteins (APRT1, PRDX2, TRM112L, PCBP2, TALDO1, ECH1, PSMA1 / 6, PRPS2, NP, TPI1, ALD, TCP1 and PDIA3) were compared to parental HEp-2 cells. It was confirmed that the expression level in HEp-2 cells was increased. On the other hand, CLIC1 and EB1 proteins were found to decrease the expression level in RR-HEp-2 cells. As can be seen from Table 1, the protein expression level of PCBP2 is 2.49 times higher than that of the parental HEp-2 cell line in RR-HEp-2 cells, whereas the CLIC1 protein is 2.05 times higher than the parental HEp-2 cell line in RR-HEp-2 cells. It can be seen that it appears low. PRDX2 is known to be an anti-oxidant protein that increases the radiation resistance of breast and head and neck cancer cells. Therefore, it can be observed that 1.86-fold expression is increased in RR-HEp-2 cells. This is indicative of a correlation with 2-D gel data and radioresistant phenotype of cells.

3-3. Protein analysis through Western analysis

Proteins showing different expression patterns were further analyzed through Western blot analysis (FIG. 3C). The experiment was conducted in the same manner as in the Western blot analysis method of Example 2-5.

As a result, the expression of CLIC1 and EB1 was reduced in RR-HEp-2 cells, while all other proteins were up-regulated in RR-HEp-2 cells. In particular, the TRM112L protein is a novel protein identified in the present invention. The mRNA expression level of this TRM112L was higher in RR-HEp-2 cells than in parental HEp-2 cells as expected (FIG. 3D). Thus protein expression of TRM112L will also be high in RR-HEp-2 cells. To confirm the results of protein genetic information between parental Hep-2 cells and radiation-resistant RR-HEp-2 cells, the same protein spots were collected from 2 of the RR-HEp-2 subtype clones (RR- # 6, 12, 13 and 18). Analyzed by comparison with -D gel map. An enlarged image of each gel of the RR-HEp-2 subtype clone isolated from HEp-2 cells was shown (FIG. 3E). Oddly, only eight protein spots in isolated subtype clones were observed to be less expressed, as compared to the parental HEp-2 cells. This showed a pattern contrary to the comparison of the results of analysis of the protein genetic information of eight protein spots in RR-HEp-2 cells. It is noteworthy that most of the identified proteins serve as modulators of the radiation resistance / stress response (Table 2).

Example 4. Functional check of CLIC1

4-1. Functional Analysis of CLIC1

We investigated the function of CLIC1 with a significant decrease in expression in radioresistant phenotype cells. The expression of spot 2 in RR-HEp-2 cells was reduced compared to the parental HEp-2 cell line (FIG. 4A), and CLIC1 was identified using MALDI-TOF-MS (FIG. 4A). CLIC1 expression was directly observed using HEp-2 cell line. Western blot and real-time PCR analysis confirmed that both protein and mRNA expression of CLIC1 were significantly reduced in RR-HEp-2 cells (FIG. 4B). These results show a rapid protein reduction of CLIC1 in RR-HEp-2 cells, which may be due to transcriptional regulation. Immunofluorescence microscopy analysis was performed to determine the intracellular location of CLIC1 in HEp-2 and RR-HEp-2 cells. CLIC1 was distributed mainly in the nucleus and was spread to low levels in the cytoplasm of normal HEp-2 cells. However, the expression of CLIC1 in RR-HEp-2 cells was found to be very small in both nucleus and cytoplasm (FIG. 4C). Interestingly, it was confirmed that the expression of CLIC1 is upregulated according to the irradiation time in parental HEp-2 cells (FIG. 4D). This confirmed that CLIC1 is a radio-reactive protein. In addition, it was observed that the expression of PARP cleaved with the induction of CLIC1 protein increased with irradiation time (FIG. 4D).

Taken together, these results suggest that CLIC1 is a negative regulator of radiation resistance obtained in laryngeal cancer cells.

4-2. CLIC1 function analysis using chemical inhibitor

In order to elucidate the role of CLIC1 in the acquired radioresistant phenotype of laryngeal cancer cells, chemical inhibitors were used to investigate the lost function in HEp-2 cells.

HEp-2 cells experienced apoptosis after 10 Gy irradiation. On the other hand, inactivation of CLIC1 by pretreatment with IAA94 (indanyloxyacetic acid-94) as a special inhibitor or DIDS (4,40-diisothiocyanatostilbene-2,20-disulfonic acid) as a general chloride channel inhibitor, markedly caused by radiation Apoptotic accumulation was blocked (Fig. 5A). NAC (N-acetyl cysteine) was used as a positive control group that inhibited radiation-induced apoptosis by reducing ROS production. Expression of cleaved PARP was induced in irradiated HEp-2 cells, and was reduced by pretreatment with the same CLIC1 chemosuppressant (FIG. 5B), showing similar cell death patterns.

In order to confirm the anti-apoptotic effect of the chemical inhibitors, colony formation assays were performed in a manner similar to that of Example 2-1 by irradiating radiation ranging from 0 to 6 Gy in HEp-2 cells. Pretreatment of IAA94 induced cell survival in HEp-2 cells as compared to the control without any treatment (FIG. 5C).

Next, direct knock-down efficiency of CLIC1 was investigated in radiation-induced apoptosis of HEp-2 cells. This was done by transient transfection of siRNA (small interfering RNA). CLIC1-specific siRNA was purchased from Bioneer, and this siRNA oligonucleotide was heated at 90 ° C. for 20 minutes to degrade into a two-dimensional structure. The sense strand and antisense strand were combined at 30 ° C. for about 1 hour. 100 nM siRNA was transfected with parent HEp-2 cells using Metafectine reagent (Biontex, M.unchen, Germany), and then incubated at 37 ° C. for 48 hours in cell growth medium. Western blot analysis showed that expression of CLIC1 was significantly suppressed by siRNA transfection (FIG. 5E). Under these circumstances, it was observed that radiation induced apoptosis of HEp-2 cells was inhibited compared to control siRNA transfected HEp-2 cells (FIG. 5D). In addition, the inhibition of CLIC1 expression using siRNA in colony formation assay showed the same result as the inhibition of CLIC1 activity using a chemical inhibitor (FIG. 5F). This suggests that inhibition of CLIC1 activity or inhibition of expression plays an important role in acquiring the radiation resistant phenotype of HEp-2 cells. Next, comparatively, the function of CLIC1 obtained in RR-HEp-2 cells but inhibited in parental HEp-2 cells was studied.

4-3. Expression Analysis of CLIC1

CLIC1 was examined through expression experiments outside the normal place. CLIC1 cDNA obtained from HEp-2 cells was digested with restriction enzymes of BamHI and XhoI and cloned into corresponding pcDNA 3.1 (+) Myc / His vector. RR-HEp-2 cells were transfected with a control vector or CLIC1 expression vector using Metafectine reagent (Biontex). Metafection-DNA complex was incubated at room temperature for 20 minutes, diluted in medium without serum, and added to cells. After culturing in growth medium for 24 hours, transfected cells were irradiated for 48 hours after irradiation, and then CLIC1 expression was observed outside the normal place.

As expected, apoptosis in response to radiation was more clearly observed in RR-HEp-2 cells transfected with myc-tagged CLIC1 expression vectors compared to cells transfected with control vectors (FIG. 6A). As shown in FIG. 6A, CLIC1 overexpression outside the normal place in RR-HEp-2 cells with the change in cell morphology promotes apoptosis very rapidly after irradiation. This was measured with a FACScan flow cytometer (FIG. 6B). Overexpression of the CLIC1 protein outside the normal place was observed by Western blot. As a result, in the overexpressed state of the same CLIC1, a clear difference in the expression amount of the cleaved PARP before and after irradiation was observed (FIG. 6C). Colony formation analysis also revealed that overexpression of CLIC1 inhibited the survival of RR-HEp-2 cells at varying amounts of radiation exposure (FIG. 6D).

Thus, it can be seen that CLIC1 contributes to the radiosensitivity of HEp-2 laryngeal cancer cells.

Example 5. Role of CLIC1

We investigated the role of CLIC1 in the development of radiation resistance in other squamous cell carcinoma cell lines. More specifically, five cancer cell lines were used. SiHa and SW756 for cervical cancer cells, NCI-H596 for liver cancer cells, and SNU-899 and SNU-1076 for laryngeal cancer cells were tested. The radiation-sensitivity of each cell line was measured by the colony formation rate in response to 4Gy radiation. In colony formation analysis, radiosensitive phenotypes were shown in SW756, NCI-H596 and SiHa cells, and radioresistant phenotypes in SNU899 and SNU1076 cells (FIG. 7A). In addition, it was observed that CLIC1 was more expressed in cell lines showing radiation sensitivity compared to cell lines showing radiation resistance. This suggests that CLIC1 expression plays an important role in radiation sensitivity in squamous cell carcinoma. Overexpression of CLIC1 outside the normal place in SNU1076 cells promotes radiation-induced apoptosis, whereas siRNA-mediated CLIC1 inhibition in SW756 cells inhibits radiation-induced apoptosis and Western blot analysis of the cleaved PARP and FACScan flow cytometer Measured through (FIGS. 7B and 7C). These results indicate that CLIC1 is strongly associated with radiosensitivity and resistance in squamous cell carcinoma cell lines.

Example 6 Association of CLIC1 with ROS Generation

Since radiation-induced ROS production plays a very important role as a mediator of apoptosis, we investigated whether radiation is involved in regulating cellular ROS levels in human HEp-2 laryngeal cancer cell lines.

10 Gy-irradiated cells were incubated with 10 μg / ml of DCF-H (2 ′, 7′-dihydrodichlorofluorescein) for 20 minutes, and then ROS were detected. Cells were recovered by trypsinization, washed three times with cold PBS, and measured by FACScan flow cytometry. Intracellular DCF-H levels were measured at 488 nm stimulation wavelength. Data analysis was performed with CellQuest (BD Biosciences, Mountain View, CA, USA) software and average fluorescence intensity.

As a result, as expected, ROS production increased significantly after parental irradiation in parental HEp-2 cells, whereas ROS levels did not change in RR-HEp-2 cells. 8A).

To determine whether CLIC1 is associated with ROS production, HEp-2 cells were irradiated with or without IAA94. As a result, when IAA94 was present and the activity of CLIC1 was suppressed, it was observed that radiation-induced ROS production was rapidly reduced (FIG. 8B). In addition, using si-RNA, it was observed that radiation-induced ROS generation was suppressed even when CLIC1 expression was directly inhibited (FIG. 8C). Next, they experimented with the CLIC1 expression vector system to investigate the association between CLIC1 and ROS production in RR-HEp-2 cells. After irradiation in RR-HEp-2 cells, CLIC1 overexpression outside the normal place led to cellular ROS accumulation (FIG. 8D).

Taken together, CLIC1 acts as a positive regulator of ROS production and modulates the radiation sensitivity of HEp-2 cells through the regulation of ROS signaling pathways.

In another experiment, we examined whether CLIC1 directly regulates the levels of the antioxidant enzymes Mn-SOD or PRDX2 in HEp-2 cells. As shown in FIGS. 8E and 8F, the regulation of CLIC1 expression by CLIC1 siRNA in HEp-2 cells or CLIC1 overexpression outside of the normal place in RR-HEp-2 cells showed that the transcription levels of Mn-SOD and PRDX2 were not irradiated. Did not change. This experiment was carried out similarly to the PCR analysis method of Example 2-2, and as a result, quantitative real-time PCR (left) and typical PCR (right) analysis.

As shown in FIG. 8C, downregulation of CLIC1 by siRNA in HEp-2 cells was found to inhibit intracellular ROS production in response to radiation. However, the transcription levels of Mn-SOD and PRDX2 were not induced, but rather, their expression was rapidly reduced (FIG. 8E). In previous data, CLIC1 overexpression outside of the normal place in RR-HEp-2 cells promoted intracellular ROS production upon irradiation (FIG. 8D), whereas the transcription levels of Mn-SOD and PRDX2 did not decrease, but rather their expression. This increase could be observed (FIG. 8F). These results indicate that the level of Mn-SOD or PRDX2 is independent of CLIC1 expression, but rather more dependent on intracellular ROS levels.

<110> Korea institute of radiological and medical sciences <120> Composition for diagnosis of radio-resistance or radio-sensitive          marker and use <130> PA100666KR <160> 18 <170> Kopatentin 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> sense primer for CLIC1 <400> 1 atggctgaag aacaaccg 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for CLIC1 <400> 2 ttatttgagg gactttga 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for Bcl-xl <400> 3 cgggcattca gtgacctgac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for Bcl-xl <400> 4 tcaggaacca gcggttgaag 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sense primer for EGFR <400> 5 cgtcgctatc aaggaattaa g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for EGFR <400> 6 tggtgggtat agattctgtg 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sense primer for GAPDH <400> 7 catctctgcc ccctctgctg a 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for GAPDH <400> 8 ggatgacctt gcccacagcc t 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for ERBB2 <400> 9 catatgtctc ccgccttctg 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for ERBB2 <400> 10 cccacacagt cacaccataa c 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer for Hsp90 <400> 11 ttggaggaac gaagaataaa gg 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for Hsp90 <400> 12 gggctctgaa ttccaactgt c 21 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> sense primer for Mn-SOD <400> 13 tgttacagcc cagatagc 18 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for Mn-SOD <400> 14 cagttacatt ctcccagttg 20 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> sense primer for PRDX2 <400> 15 aaagggaagt acgtggtc 18 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for PRDX2 <400> 16 gggcttaatc gtgtcactg 19 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> sense primer for TRM112-like protein <400> 17 cttacccaca atctgct 17 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for TRM112-like protein <400> 18 gaacatacgt ccagattcc 19

Claims (14)

CLC1 (Chloride intracellular channel protein 1, NCBI GenBank Accession No. NM_001288) A composition for diagnosing radiation resistance or sensitivity of cancer cells comprising an agent for measuring the level of mRNA of the gene or a protein thereof.
According to claim 1, wherein the composition is APRT1 (Adenine phosphoribosyltransferase, NCBI GenBank Accession No. NM_000485), PRDX2 (Peroxiredoxin-II, NCBI GenBank Accession No. NM_005809), TRM112L (TRM112-like protein, NCBI GenBank Accession No. NM_016404) , PCBP2 (Poly (rC) -binding protein 2, NCBI GenBank Accession No. NM_001098620), TALDO1 (Transaldolase-1, NCBI GenBank Accession No. NM_006755), ECH1 (Enoyl Coenzyme A hydratase 1, NCBI GenBank Accession No. NM _001398) , PSMA1 (Proteasome subunit alpha type-1, NCBI GenBank Accession No. NM_148976), PRPS2 (Phosphoribosyl pyrophosphate synthetase 2, NCBI GenBank Accession No. NM_001039091), NP (Nucleoside phosphorylase, NCBI GenBank Accession No. NM_000270Pro), PSMA6 subunit alpha type-6, NCBI GenBank Accession No. NM_002791), TPI1 (Triosephosphate isomerase, NCBI GenBank Accession No. NM_000365), ALD (Aldehyde reductase, NCBI GenBank Accession No. NM_006066), EB1 (End-binding protein 1, NCBI GenBank Accession No. NM_012325), TCP1 (T-complex 1, NCBI GenBank Accession No. NM_030752) and PDIA3 (Protein disulfide-isomerase A3, NCBI GenBank Accession No. NM_005313) radiation resistance or sensitivity of cancer cells further comprising an agent for measuring the level of mRNA or protein of one or more genes selected from the group Diagnostic composition.
The composition for diagnosing radiation resistance or sensitivity of cancer cells according to claim 1, wherein the cancer is head and neck cancer, laryngeal cancer, cervical cancer or colorectal cancer.
The composition for diagnosing radiation resistance or sensitivity of cancer cells according to claim 1 or 2, wherein the agent for measuring the level of the gene mRNA comprises a primer specifically binding to the gene.
The composition for diagnosing radiation resistance or sensitivity of cancer cells according to claim 1 or 2, wherein the agent for measuring the protein level of the gene comprises an antibody specific for the protein.
A kit for diagnosing radiation resistance or sensitivity of cancer cells comprising the composition of claim 1.
The kit for diagnosing radiation resistance or sensitivity of cancer cells according to claim 6, wherein the kit is an RT-PCR kit, a DNA chip kit, a microarray, and a protein chip kit.
Measuring mRNA levels from isolated biological samples using primers that specifically bind to the CLIC1 gene; And
Comparing the change in the mRNA level with the mRNA level of the normal control sample, the method of providing information for diagnosing radiological resistance or sensitivity of cancer patients.
The method according to claim 8, wherein the Adenine phosphoribosyltransferase (APRT1), NCBI GenBank Accession No. NM_000485), PRDX2 (Peroxiredoxin-II, NCBI GenBank Accession No. NM_005809), TRM112L (TRM112-like protein, NCBI GenBank Accession No. NM_016404), PCBP2 Poly (rC) -binding protein 2, NCBI GenBank Accession No. NM_001098620), TALDO1 (Transaldolase-1, NCBI GenBank Accession No. NM_006755), ECH1 (Enoyl Coenzyme A hydratase 1, NCBI GenBank Accession No. NM _001398), PSMA1 ( Proteasome subunit alpha type-1, NCBI GenBank Accession No. NM_148976), PRPS2 (Phosphoribosyl pyrophosphate synthetase 2, NCBI GenBank Accession No. NM_001039091), NP (Nucleoside phosphorylase, NCBI GenBank Accession No. NM_000270), PSMAunit subtype 6, NCBI GenBank Accession No. NM_002791), TPI1 (Triosephosphate isomerase, NCBI GenBank Accession No. NM_000365), ALD (Aldehyde reductase, NCBI GenBank Accession No. NM_006066), EB1 (End-binding protein 1, NCBI GenBank Accession325 No. NM_002791) ), TCP1 (T-complex 1, NCBI GenBank Accessi on No. NM_030752) and PDIA3 (Protein disulfide-isomerase A3, NCBI GenBank Accession No. NM_005313), further comprising measuring and comparing the mRNA level of one or more genes selected from the group consisting of a method for providing information for diagnosing radiation resistance or sensitivity of cancer patients.
Contacting an antibody specific for a protein of CLIC1 with an isolated biological sample to determine protein levels by antigen-antibody complex formation; And
Comparing the change in the amount of protein formation with the protein level of the normal control sample, Method of providing information for diagnosing radiation resistance or sensitivity of cancer patients.
The method according to claim 10, comprising: Adenine phosphoribosyltransferase (APRT1), NCBI GenBank Accession No. NM_000485), PRDX2 (Peroxiredoxin-II, NCBI GenBank Accession No. NM_005809), TRM112L (TRM112-like protein, NCBI GenBank Accession No. NM_016404), PCBP2 Poly (rC) -binding protein 2, NCBI GenBank Accession No. NM_001098620), TALDO1 (Transaldolase-1, NCBI GenBank Accession No. NM_006755), ECH1 (Enoyl Coenzyme A hydratase 1, NCBI GenBank Accession No. NM _001398), PSMA1 ( Proteasome subunit alpha type-1, NCBI GenBank Accession No. NM_148976), PRPS2 (Phosphoribosyl pyrophosphate synthetase 2, NCBI GenBank Accession No. NM_001039091), NP (Nucleoside phosphorylase, NCBI GenBank Accession No. NM_000270), PSMAunit subtype 6, NCBI GenBank Accession No. NM_002791), TPI1 (Triosephosphate isomerase, NCBI GenBank Accession No. NM_000365), ALD (Aldehyde reductase, NCBI GenBank Accession No. NM_006066), EB1 (End-binding protein 1, NCBI GenBank Accession325 No. NM_002791) ), TCP1 (T-complex 1, NCBI GenBank Access ion No. NM_030752) and PDIA3 (Protein disulfide-isomerase A3, NCBI GenBank Accession No. NM_005313), further comprising the step of measuring and comparing the protein level of one or more genes selected from the group of information for providing information for diagnosing radiation resistance or sensitivity of cancer patients.
In order to provide the information necessary for predicting the prognosis of the radiation therapy of cancer patients, a method for detecting a radiation resistance or sensitivity diagnostic marker by comparing the level of the mRNA or protein of CLIC1 with the expression level of normal cells from a sample of patients receiving radiation therapy .
The method according to claim 12, wherein the Adenine phosphoribosyltransferase (APRT1), NCBI GenBank Accession No. NM_000485), PRDX2 (Peroxiredoxin-II, NCBI GenBank Accession No. NM_005809), TRM112L (TRM112-like protein, NCBI GenBank Accession No. NM_016404), PCBP2 Poly (rC) -binding protein 2, NCBI GenBank Accession No. NM_001098620), TALDO1 (Transaldolase-1, NCBI GenBank Accession No. NM_006755), ECH1 (Enoyl Coenzyme A hydratase 1, NCBI GenBank Accession No. NM _001398), PSMA1 ( Proteasome subunit alpha type-1, NCBI GenBank Accession No. NM_148976), PRPS2 (Phosphoribosyl pyrophosphate synthetase 2, NCBI GenBank Accession No. NM_001039091), NP (Nucleoside phosphorylase, NCBI GenBank Accession No. NM_000270), PSMAunit subtype 6, NCBI GenBank Accession No. NM_002791), TPI1 (Triosephosphate isomerase, NCBI GenBank Accession No. NM_000365), ALD (Aldehyde reductase, NCBI GenBank Accession No. NM_006066), EB1 (End-binding protein 1, NCBI GenBank Accession325 No. NM_002791) ), TCP1 (T-complex 1, NCBI GenBank Access ion No. NM_030752) and PDIA3 (Protein disulfide-isomerase A3, NCBI GenBank Accession No. NM_005313), further comprising the step of measuring and comparing the mRNA or protein level of one or more genes selected from the group consisting of.
The method of claim 12 or 13, wherein the cancer is head and neck cancer, laryngeal cancer, cervical cancer, or colorectal cancer.

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