KR20100085315A - A method for protecting dna damage using pig3 - Google Patents

Abstract

PURPOSE: A composition and method for protecting DNA damage using PIG3 are provided to suppress cytotoxicity and to relate to normal recognition in DNA damage. CONSTITUTION: An anti-cancer aid contains an inhibitor for expression or activation of PIG3(p53-inducible gene 3). The PIG3 has an amino acid sequence of sequence number 1. The inhibitor for expression of PIG3 is an antisense nucleotide which complementarily binds to mRNA of PIG3 gene, small interfering RNA(siRNA) or small hairpin RNA(shRNA). The inhibitor for expression or activation of PIG3 treats and prevents cancer by administering to cancer cells. A cosmetic composition for anti-aging contains PIG3 gene construct as an active ingredient. The PIG3 gene has a nucleotide sequence of sequence number 2.

Description

A method for protecting DNA damage using PIG3}

The present invention relates to a method for protecting DNA damage using PIG3, and more particularly, PIG3 (p53-inducible gene 3) is involved in the response pathway to DNA damage caused by DNA damaging agents, and The present invention relates to protection of DNA damage by using a direct role in signaling by DNA damage.

DNA damage is known to cause many diseases, including cancer and aging. To overcome this, a system exists that recognizes DNA damage and stops the cell cycle and repairs it. However, it has been observed that in many diseased cells the system does not operate normally. Therefore, it is very important to prevent and treat diseases related to DNA mutations by ensuring that DNA damage response works normally after DNA damage and that these damages are repaired without mutation.

DNA damage responses are complex signaling pathways that involve the organization of events in various cells that activate rapidly in response to DNA damage. These signaling pathways inhibit many cell cycles, promote DNA repair (called the DNA damage checkpoint), and many factors that cause cell death if DNA damage is too much to be repaired. (Harper, JW and Elledge, SJ, Mol Cell , 28, 739-745, 2007; Bartek, J. and Lukas, J., Curr Opin Cell Biol , 19, 238-245, 2007).

In mammalian cells, the phosphatidylinositol 3-kinase-like kinase (PIKK) family, ataxia telangiectasia mutated (ATM), ATM and Rad3 related proteins (ATM and Rad3 related) ATR), and DNA dependent protein kinases (DNA-PKs) play an important role in recognizing and responding to chromosomal damage (Shiloh, Y., Curr Opin Genet Dev , 11, 71-77, 2001). Falck, J., Coates, J. and Jackson, SP, Nature , 434, 605-611, 2005).

The DNA damage response is a linear process in which an initial damage signal initiated by a sensor is transmitted to mediators and transducers, and the mediator and transmitter transforms the signal to many effectors. It is considered to be made. Disruption of the DNA damage response pathway in human cells results in increased risk of genetic instability and cancer progression (Kastan, MB and Bartek, J., Nature , 432, 316-323, 2004; Zhou, BB and Elledge, SJ, Nature , 408, 433-439, 2000). Therefore, it is necessary to understand the complex mechanism at the molecular level for knowledge of cancer progression and treatment.

In the last few years, many researchers have studied how DNA damage signals are integrated in a cellular response to DNA damage, but have initiated an early event that is stimulated by DNA damage that precedes the spread of intracellular DNA destruction signals. Little is known about the mechanism.

p53 inducible gene 3 ( PIG3 ) has been identified through analysis of gene expression in studies to confirm that it is a gene induced by p53 before initiation of apoptosis (Polyak, K., et al., Nature , 389, 300-305, 1997). p53 interacts with the pentanucleotide microsatellite sequence in the PIG3 promoter [(TGYCC) n, where Y = C or T], required for the transcriptional activity of the PIG3 promoter by p53 (Contente, A., et al., Nat Genet , 30, 315-320, 2002). The amino acid sequence of PIG3 shows significant homology with the amino acid sequence of NADH quinine oxidoreductase1 (NQO1), whereby PIG3 is similar to NQO1 and PIG3 is the generation of reactive oxygen species (ROS). (Polyak, K., et al., Nature , 389, 300-305, 1997) and have been shown to be important downstream mediators of p53 dependent apoptosis responses.

In addition, human cellular apoptosis susceptibility protein (hCAS / CSE1L) interacts with the PIG3 promoter and affects p53 dependent apoptosis by regulating PIG3 expression (Tanaka, T., et al., Cell , 130, 638-650, 2007). However, since expression of PIG3 alone is insufficient to induce apoptosis (Polyak, K., et al., Nature , 389, 300-305, 1997), it is presumed that several factors that cause apoptosis cooperate.

UV irradiation, on the other hand, induces alternative splicing of PIG3 pre-mRNAs to produce splice variant proteins that are rapidly degraded by the proteasome degradation pathway, which damages the DNA of the cell. There is a new legend between the regulation of reaction and alternative splicing (Nicholls, CD, et al., J Biol Chem , 279, 24171-24178, 2004). In addition, heterogeneous nuclear ribonuclear proteins (hnRNPs) A1 and A2 contribute to normal replacement splicing of PIG3 that is not related to UV-induced replacement splicing, which is normal and UV-induced replacement of PIG3. It suggests that the mechanism differs from the mechanism of mediating flies (Nicholls, CD and Beattie, TL, Biochim Biophys Acta , 1779, 838-849, 2008). PIG3 is therefore considered to modulate unknown functions by cooperation of DNA damage response pathways.

Cell cycle checkpoints, which cause cell cycles to pause during DNA damage, occur. These phenomena occur to prevent gene mutations at the damaged site, and play a very important role in gene safety. Cell cycle checkpoints in DNA damage include G2 / M checkpoints (which stop the cell cycle just before the cell is divided into two) and intra-S checkpoints that stop when the DNA is divided into two.

DNA checkpoint activation integrates signals that regulate DNA damage responses including DNA damage repair, cell cycle arrest and cell death (Abraham, RT, Genes Dev , 15, 2177-2196, 2001; Shiloh , Y. Nat Rev Cancer , 3, 155-168, 2003). Intra-S stage checkpoints serve to inhibit spontaneous replication-fork disruption and intra S-phase progression after DNA damage (Bartek, J., et al., Nat Rev Mol Cell Biol , 5 , 792-804, 2004). The G2 / M checkpoint activates to inhibit the progression of mitosis by cells with damaged or incomplete DNA. Cells that fail to activate the intra S-phase checkpoint can inhibit progression to mitosis by activating the G2 / M checkpoint (Sancar, A., et al., Annu Rev Biochem , 73, 39-85, 2004 ).

The activity of ATM and ATR by damaged DNA results in direct or indirect phosphorylation or activity of many downstream targets. Checkpoint kinases Chk1 and Chk2 play important roles in cell cycle regulation (Su, TT, Annu Rev Genet , 40 , 187-208, 2006; Bartek, J., et al., Nat Rev Mol Cell Biol , 2 , 877-886, 2001). Chk1 and Chk2 are phosphorylated at serine 345 and threonine 68 by gene toxicity exposure, respectively. The disruption of Chk1 or Chk2 causes loss of intra-S and G2 / M checkpoints, indicating the role of Chk1 and Chk2 in response to DNA damage. The ATM / Chk2 pathway mainly responds to DSB induced by irradiation, while the ATM / Chk2 pathway is activated by large amounts of DNA damage following UV irradiation or replication breakdown (Zhou, BB, Nature , 408, 433). -439, 2000).

In response to DNA damage, H2AX is phosphorylated at Ser139 (γ-H2AX) and is dispersed and located at the site of DNA damage (Su, TT, et al., Annu Rev Genet , 40, 187-208, 2006). PIKK's ability to phosphorylated H2AX in response to DNA damage requires the proper location of recovery mechanisms. Thus, phosphorylation of H2AX is an indicator of whether the DNA damage response pathway is activated in response to DNA damage stimuli (Ward, IM et al., J Biol Chem , 276, 47759-47762, 2001; Rogakou, EP, et al. , J Biol Chem , 273, 5858-5868, 1998).

Various DNA damage signal proteins are targets for ATM / ATR-mediated phosphorylation and are involved in the transmission of DNA damage signals to downstream targets. After DNA damage, the proteins gather at the site of DNA damage, and each protein forms intranuclear aggregates induced by DNA damage. The order and timing of these events are considered important for checkpoint response and DNA recovery (Stewart, GS, et al., Nature , 421, 961-966, 2003).

Therefore, while we are studying the molecular mechanism by which PIG3 is involved in cellular response to DNA damage, knockdown of PIG3 increases the cell's sensitivity to UV and radiomimetic drugs, and Defects in the intra-S phase and G2 / M checkpoints in response to damage, unstable induction of Chk1 and Chk2 phosphorylation following DNA damage, unstable H2AX phosphorylation in response to DNA damage And by confirming that γ-H2AX aggregation is inhibited, the present invention was completed by discovering that PIG3 functions as an upstream component of the DNA damage pathway important for the activity and maintenance of the DNA damage suppression signaling pathway.

It is an object of the present invention to provide a method of increasing cell resistance by protecting DNA damage against harmful substances causing DNA damage such as radiation or ultraviolet light using PIG3 (p53-inducible gene 3).

In order to achieve the above object, the present invention provides a cancer treatment aid containing an inhibitor of the expression or activity of PIG3 (p53-inducible gene 3).

The present invention also provides a method for preventing or treating cancer, comprising administering an inhibitor of PIG3 expression or activity to cancer cells.

The present invention also provides an anti-aging cosmetic composition containing PIG3 as an active ingredient.

The present invention also provides an anti-aging cosmetic composition containing the PIG3 gene construct as an active ingredient.

In addition, the present invention provides a method of increasing the resistance of a cell to DNA damaging agents, comprising administering to the cell a composition containing PIG3 or PIG3 gene construct as an active ingredient.

DNA damage protection composition containing PIG3 of the present invention as an active ingredient inhibits cytotoxicity caused by ultraviolet (UV) or radiation-like drugs, is involved in the normal operation of the cell cycle check system (cell cycle ckeckpoint), DNA DNA damage sensors that recognize damage can contribute to the survival and gene safety of cells by increasing cell resistance to harmful substances that cause DNA damage, such as radiation or ultraviolet rays.

Hereinafter, the present invention will be described in detail.

The present invention provides cancer treatment aids containing inhibitors of the expression or activity of PIG3.

The PIG3 preferably has an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto.

The inhibitor of expression of PIG3 is preferably an antisense nucleotide, small interfering RNA (siRNA) or small hairpin RNA (shRNA) that complementarily binds to mRNA of the PIG3 ( p53-inducible gene 3 ) gene. In addition, the activity inhibitor of PIG3 is preferably any one selected from the group consisting of compounds, peptides, peptide mimetics and antibodies that bind to PIG3, but is not limited thereto.

The cancer is preferably one selected from the group consisting of rectal cancer, colon cancer, colon cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer and liver cancer, but is not limited thereto.

The present inventors prepared a PIG3 knockdown cancer cells using PIG3 siRNA to determine the relationship between the DNA damage response of PIG, and then analyzed the cell viability of the cells for sensitivity to DNA damage factors such as UV or radioactive substances. survival assay). As a result, PIG3 knockdown cells showed higher sensitivity to DNA damaging factors than control cells (see FIGS. 1B and 1C).

In addition, the present inventors have performed DNA double strand cleavage using Pulsed-Field Gel Electrophoresis (PFGE) to determine whether increased sensitivity to DNA damaging factors of PIG3 knockdown cells affects defects in DNA damage repair. The degree of retention of (DNA double-strand break; DSB) was confirmed. As a result, PIG3 knockdown cells maintained DSB at a higher rate than control cells (see FIG. 2).

Thus, it can be seen that PIG3 increases the cell's sensitivity to DNA damage factors and is involved in repairing DNA damage.

In addition, the present inventors have found that PIG3 is involved in the regulation of intra-S checkpoint and G2 / M checkpoint following DNA damage. After treatment of the damaging factor, the degree of DNA synthesis inhibition was analyzed using the BrdU incorporation assay and G2 / M checkpoint analysis to analyze the cell cycle checkpoint activity. As a result, control cells showed a significant degree of DNA synthesis inhibition and stopped at the G2 stage, whereas PIG3 knockdown cells efficiently inhibited intra-S phase checkpoint activity, indicating a low level of DNA synthesis inhibition and a G2 stage. Mitosis proceeded without stopping at (see FIGS. 3A-3C).

Thus, it can be seen that PIG3 is involved in cell cycle checkpoint activity that occurs when DNA damages.

In addition, the present inventors treated with DNA damaging factors in the control and PIG3 knockdown cancer cells to determine whether PIG3 is involved in the regulation of Chk1 / Chk2 signaling pathways, and immunoprecipitation assay and Confirmed using Western blot analysis. As a result, PIG3 knockdown cells inhibited the degree of phosphorylation of Chk2 at Thr68, an active site, in response to DNA damaging factors, and suppressed a sharp decrease in the level of Cdc25A expression and decreased Chk1 Ser345 phosphorylation in response to DNA damage factors (FIG. 4A). To FIG. 4C).

Thus, it can be seen that PIG3 regulates Chk1 / Chk2 in response to DNA damage. Since phosphorylation of Chk2 and Chk1 is known as an important mediator of cell cycle checkpoints that occur after DNA damage, it can be seen that PIG3 phosphorylates Chk1 and Chk2 in response to DNA damage to operate the cell cycle checkpoint normally.

In addition, the present inventors treated the DNA damage factor in the control and PIG3 knockdown cancer cells to determine whether PIG3 is involved in the DNA damage signal by H2AX protein, and then immunofluorescence microscopy and Western blot analysis using immunofluorescence microscopy The formation of -H2AX foci and the degree of phosphorylation of H2AX were confirmed. As a result, PIG3 knockdown cells significantly reduced the degree of aggregation of γ-H2AX and the degree of H2AX phosphorylation in response to DNA damaging factors compared to control cells (see FIGS. 5A-5D).

Thus, it can be seen that PIG3 is involved in the DNA damage signal for the H2AX protein and that PIG3 is involved in the signal that causes DNA repair as well as the activity of the checkpoint in response to DNA damage factors.

In addition, the present inventors prepared the nuclear and cytosolic fractions after treatment of DNA damage factors in cancer cells to determine the intracellular location of PIG3 after DNA damage, and then analyzed the degree of PIG3 expression using Western blot analysis in each fraction. Analyzed. As a result, PIG3 was mainly located in the cytoplasmic fraction but about 30% was in the nucleus (see FIG. 6A). In addition, expression constructs encoding the full-length (GFP-tagged full-length), C-terminal (1-480 nt) deficient mutations, and N-terminal (421-999 nt) deficient mutations of PIG3 were prepared (Fig. 6B) After each transformed with cancer cells, PIG3 expression levels were analyzed by Western blotting. As a result, it was confirmed that the N-terminal region (1-480 nt) of PIG3 is an essential region for the intranuclear position (see FIG. 6C).

Thus, it can be seen that PIG3 is located in the nucleus by the N-terminal site and that PIG3 in the nucleus is involved in the DNA damage signal.

In addition, the present inventors have used immunofluorescence staining after treatment of DNA damaging factors in cancer cells to determine the extent and location of PIG3 in the nucleus aggregate formation and the association of DNA damage signal proteins in response to DNA damage. And analyzed. As a result, PIG3 aggregates were dispersed in the nucleus and formed high levels of aggregates at DNA break sites in response to DNA damage, and the PIG3 aggregates were co-located with γ-H2AX and 53BP1 aggregates. It was confirmed that there is a bond with (see Fig. 7a to 7d).

Thus, PIG3 aggregation induced by DNA damage represents a site of DSB processing, it can be seen that PIG3 is upstream of the DNA damage signal, and PIG3 acts on a damage sensor system for DNA damage to monitor normal DNA damage. It can be seen that it is involved in this.

We also knocked down the cells treated with PIKK inhibitors, ATM, ATR and DNA-PK to determine if PIG3 was related to PIKK, ATM, ATR, DNA-PK and p53 in response to DNA damage. After processing the DNA damaging factor in the cells and p53 ^{− / −} cells, the degree of PIG3 expression was confirmed by Western blot analysis. As a result, aggregates of PIG3 decreased expression by PIKK inhibitors, and there was no significant difference in expression by knockdown of ATM, ATR and DNA-PK, respectively, and formed high aggregates of PIG3 in p53 ^{− / −} cells. (See FIGS. 8A-8E).

Thus, it can be seen that PIG3 aggregate formation is involved by one or more of the PIKK families and is dependent on p53, and that PIG3 is directly involved in DNA damage signaling.

Through these results, inhibition of the expression or activity of PIG3 increases the cell's sensitivity to DNA damaging factors such as UV or radioactive drugs, and can detect defects of the Intra-S phase and G2 / M checkpoints in response to DNA damage. Efficient induction of Chk1 and Chk2 phosphorylation due to DNA damage becomes unstable, inhibits H2AX phosphorylation and γ-H2AX aggregation in response to DNA damage, and inhibits signal transduction of Chk1 / Chk2 in response to DNA damage By causing pathway defects, cancer cells may be less resistant to DNA damage.

Therefore, it can be seen that the PIG3 expression or activity inhibitor of the present invention can enhance the death of cancer cells in the treatment of DNA damage factors such as radiation or anticancer agents in the treatment of cancer cells.

The cancer treatment aid of the present invention may contain one or more active ingredients that exhibit the same or similar function in addition to the expression or activity inhibitor of PIG3.

The cancer treatment aid of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as needed. And other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions, and the like, specifically for target organs. Target organ specific antibodies or other ligands can be used in combination with the carrier so that they can act. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

The nucleotides or nucleic acids used in the present invention can be prepared for the purpose of topical, parenteral, nasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal and the like. Preferably, the nucleic acid or vector is used in injectable form. Thus, in particular, the area to be treated may be mixed with any pharmaceutically acceptable vehicle for injectable compositions for direct infusion. Adjuvants of the present invention may in particular comprise isotonic sterile solutions or dry lyophilized compositions which allow the composition of injectable solutions upon addition of sterile water or appropriate physiological saline. Direct injection of nucleic acid into a patient's tumor is advantageous because it allows the treatment efficiency to be focused on the infected tissue. The dosage of nucleic acid used can be adjusted by various parameters, in particular by gene, vector, mode of administration used, disease in question or alternatively desired duration of treatment. In addition, the range varies depending on the weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of the patient. The daily dosage is about 0.001 to 10.0 mg / kg, preferably 0.01 to 1.0 mg / kg, preferably administered once to several times a day.

The present invention also provides a method for preventing or treating cancer, comprising administering an inhibitor of PIG3 expression or activity to cancer cells.

The PIG3 preferably has an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto.

The inhibitor of expression of PIG3 is preferably an antisense nucleotide, small interfering RNA (siRNA) or small hairpin RNA (shRNA) that complementarily binds to mRNA of the PIG3 ( p53-inducible gene 3 ) gene. In addition, the activity inhibitor of PIG3 is preferably any one selected from the group consisting of compounds, peptides, peptide mimetics and antibodies that bind to PIG3, but is not limited thereto.

The cancer is preferably one selected from the group consisting of rectal cancer, colon cancer, colon cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer and liver cancer, but is not limited thereto.

Administration of inhibitors of PIG3 expression or activity to cancer cells increases the cell's sensitivity to DNA damaging factors, such as UV or radioactive drugs, and can result in defects of the Intra-S phase and G2 / M checkpoints in response to DNA damage. Efficient induction of Chk1 and Chk2 phosphorylation due to DNA damage becomes unstable, inhibits H2AX phosphorylation and γ-H2AX aggregation in response to DNA damage, and inhibits signal transduction of Chk1 / Chk2 in response to DNA damage By causing pathway defects, cancer cells may be less resistant to DNA damage.

The present invention provides an anti-aging cosmetic composition containing PIG3 (p53-inducible gene 3) as an active ingredient.

The PIG3 preferably has an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto.

The present invention also provides an anti-aging cosmetic composition containing PIG3 ( p53-inducible gene 3 ) gene construct as an active ingredient.

The PIG3 gene preferably has a nucleotide sequence set forth in SEQ ID NO: 2, but is not limited thereto.

PIG3 inhibits the cell's sensitivity to DNA damaging agents, such as ultraviolet (UV) or radiomimetic drugs, is involved in repairing DNA damage, and plays a role in the activity of the DNA damage checkpoint pathway. PIG3 is an important component of the DNA damage response pathway and DNA damage by being involved, forming intranuclear aggregates in response to DNA damage, involved in phosphorylation of Chk1, Chk2 and H2AX, and acting downstream of ATM and ATR. Since it plays a direct role in signal transduction, it can be seen that it can be useful for preventing aging by protecting DNA damage.

When the PIG3 of the present invention is used as a cosmetic composition, the cosmetic composition prepared by containing the PIG3 as an active ingredient may be prepared in the form of a general emulsion formulation and a solubilized formulation. Cosmetics of the emulsified formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations are flexible cosmetics.

Suitable cosmetic formulations include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), nonionic vesicles obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase, for example. It may be provided in the form of a dispersant, cream, skin, lotion, powder, ointment, spray or cone stick. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the cosmetic composition, in addition to the PIG3 of the present invention, fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water Commonly used in ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic field, such as any other ingredient.

In addition, the present invention provides a method for treating DNA damage factors (DNA damaging agents) comprising the step of administering to the cell a DNA damage protection composition containing PIG3 (p53-inducible gene 3) or PIG3 gene construct as an active ingredient. It provides a method of increasing resistance.

The PIG3 preferably has an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto.

The PIG3 gene preferably has a nucleotide sequence set forth in SEQ ID NO: 2, but is not limited thereto.

The DNA damage factor is preferably ultraviolet (UV) or radiomimetic drug (radiomimetic drug), the radiopharmaceutical drug is preferably bleomycin (bleomycin) or neocardinostatin (neocarzinostatin), but is not limited thereto.

PIG3 of the present invention inhibits the cell's sensitivity to DNA damage factors such as ultraviolet or radioactive drugs, is involved in repairing DNA damage, is involved in the activity of the DNA damage checkpoint pathway, and responds to DNA damage As a result, they form intranuclear aggregates, participate in the phosphorylation of Chk1, Chk2 and H2AX, and function downstream of ATM and ATR, indicating that they are important components of the DNA damage response pathway and play a direct role in DNA damage signaling. .

Therefore, it can be seen that the composition for protecting DNA damage containing PIG3 as an active ingredient may contribute to cell survival and gene safety by increasing cell resistance to harmful substances causing DNA damage.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples specifically illustrate the present invention, and the content of the present invention is not limited to Examples, Experimental Examples, and Preparation Examples.

Example 1 Cell Culture and DNA Damage Factor Treatment

We used HCT116 p53 ^{+ / +} and p53 ^-/- colorectal carcinoma cells, 10% heat inactivated fetal bovine serum (Cambrex Corp., Walkersville, MD), 100 units / ml. In a 5% CO ₂ wet incubator in Iscove's modified Dulbecco's medium (IMDM; Gibco-BRL, Grand Island, NY) with phenylsilin and 100 μg / ml streptomycin sulfate (Invitrogen, Carlsbad, Calif.). Incubated at 37 ° C. HeLa cervix adenocarcinoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL).

Radioactive drugs bleomycin (Sigma, St. Louis, Mo.) and neocarzinostatin (Sigma) were added to the cell medium to a final concentration of 5 mU and 200 ng / ml, respectively. The UV treatment washed the cells and then exposed the UV at a 10 J / m ² irradiation dose using a 254-nm UVC lamp (UVP; Model UVGL-25) in a minimal volume of serum free medium. UV exposed cells were incubated in medium containing serum at 37 ° C.

Example 2 Preparation of PIG3 Knockdown Cells

In order to knock down PIG3 expression, we found a small interference from p53 inducible protein 3 (TP53I3; PIG3) mRNA sequence (GenBank accession number NM_004881), a human tumor protein from NCBI Entrez nucleotide database. Small interfering RNA (siRNA) target sites were selected. To determine if the target site was specific for human PIG3, the target site was examined using BLAST search (National Center for Biotechnology Information). The sequences of sense and antisense RNA consisting of 21 nucleotides are as follows: 5'-AAAUGUUCAGGCUGGAGACUAdTdT-3 '(SEQ ID NO: 3) and 5'-UAGUCUCCAGCCUGAACAUUUdTdT-3' (SEQ ID NO: 4). The sequence of the negative control siRNA duplex (Bioneer company, Korea) is as follows: 5'-CCUACGCCAAUUUCGUdTdT-3 '(SEQ ID NO: 5) and 5'-ACGAAAUUGGUGGCGUAGGdTdT-3' (SEQ ID NO: 6). The siRNA duplexes were transformed into cells using RNAiMAX (Invitrogen) according to the manufacturer's instructions. These shRNAs were prepared by transcription-based methods using the pSilencer ™ hygro kit (Ambion, Austin, TX) according to the manufacturer's instructions. The cells were transformed with Lipofectamine 2000 (Invitrogen) using a shRNA expression plasmid based on the pSilencer hygro vector (Ambion) prepared above containing a human U6 promoter and a hygromycin resistance gene. For stable knockdown of PIG3, 300 μg / ml hygromycin resistant colonies were selected after transformation with psilencer-empty or psilencer-PIG3 vectors. ATM, ATR and DNA-PK siRNA duplexes were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Example 3 Preparation of Cells Comprising Full Length PIG3, N-Terminal Deficient PIG3, and C-terminal Deficient PIG3

To prepare full-length, N-terminal deficiency and C-terminal deficiency of PIG3, each site of human PIG3 cDNA was amplified from human fibroblast GM00637 by RT-PCR using PIG3 oligo primers of the following sequence: full length PIG3 Sense, 5'-AAGGAAATAACCACCATGTTAGCCGTGCAC-3 '(SEQ ID NO: 7) and antisense 5'-CTGGGGCAGTTCCAGGACGATCTT-3' (SEQ ID NO: 8) for preparation; Sense, 5'-AAGGAAATAACCACCATGTTAGCCGTGCAC-3 '(SEQ ID NO: 9) and antisense 5'-GAGTTGGATAGCAGCTGTGCCCACA-3' (SEQ ID NO: 10) for preparation of C-terminal deficient PIG3; Sense, 5′-AAGGAAARAACCACCATGGGAGACTATGTGCTA-3 ′ (SEQ ID NO: 11) and antisense 5′-CTGGGGCAGTTCCAGGACGATCTT-3 ′ (SEQ ID NO: 12) for preparation of N-terminal deficient PIG3. The amplified PIG3 PCR product was cloned into a pCR8 / GW / TOPO vector (invitrogen) and then contained a green fluorescence protein (GFP) using the LR reaction by Gateway LR clonase ™ II Enzyme Mix (invitrogen). The mammalian expression pcDNA-DEST47 Gateway ^® vector was inserted. PIG3 sequence and orientation were confirmed by automated DNA sequencing. The vector was transformed into cells using LipofectAMINE 2000 (Invitrogen) into the appropriate plasmid according to the manufacturer's instructions.

Experimental Example 1 Investigation of the Expression of PIG3 in PIG3 Knockdown Cells

In order to investigate the DNA damage response of PIG3, the inventors prepared HCT116 and HeLa cell lines containing PIG3-targeted small hairpin RNA (shRNA) expression vectors, followed by immunoprecipitation assay and Western assay. Western blot analysis was used to confirm the expression level of PIG3.

Specifically, immunoprecipitation assay and Western blot analysis were performed as follows: Cells were prepared in RIPA buffer [50 mM Tris-HCl [pH 7.5], 150 containing a fortease inhibitor (Roche Diagnostic Corp., Indianapolis, IN). mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) or M-PER Buffer Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Equal amounts of protein were separated by 6-15% SDS-PAGE by electrotransfer into polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.). The membrane was blocked for 1 hour with TBS-t (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.1% tween-20) containing 5% non-fat milk, followed by primary antibody. Incubated at room temperature. The block was washed four times for 15 minutes with 0.1% Tween 20 containing TBS-t followed by a peroxidase-binding secondary antibody (1: 5000, Jackson ImmunoResearch Inc., West Grove, PA). Incubated for 1 hour. The membrane was washed four times and then developed using a chemiluminescence detection system (ECL; GE Healthcare, Buckinghamshire, UK). For immunoprecipitation assays, RIPA extracts were pre-cleared with protein A-sepharose beads (GE Healthcare.) And then incubated overnight at 4 ° C. with appropriate antibodies. After the addition of Protein A-Sepharose beads, the reaction was incubated for 3 hours at 4 ° C. in circulation. The beads were washed five times with RIPA buffer without protease inhibitors, then resuspended in SDS sample buffer and then boiled for 5 minutes. The samples were then analyzed by western blotting with appropriate antibodies. At this time, the PIG3 protein was detected by a rabbit polyclonal PIG3 antibody (H300, Santa Cruz Biotechnology) in a 1: 1000 dilution. Hereinafter, the following antibodies were used for Western blot analysis: anti-αtubulin mAb (TU-02, 1: 5000), anti-Cdc25A mAb (1: 500), anti-myc mAb (1: 500, Santa cruz ); Anti-Chk1 pAb (1: 1000), anti-Chk1-p (S345) pAb (1: 1000), anti-Chk2 pAb (1: 1000), anti-Chk2-p (T68) pAb (1: 1000, Cell Signaling Technology).

As a result, HCT116 and HeLa cell lines stably transformed with PIG3 shRNA showed a 90% or more reduction in PIG3 expression compared to scrambled (control) shRNA transformed cells (FIG. 1A).

Experimental Example 2 Investigation of Sensitivity to DNA Damage Factors in PIG3 Knockdown Cells

In order to investigate the response to DNA damaging reagents in control shRNA and PIG3 shRNA expressing cells, the present inventors treated the control and PIG3 knockdown HCT116 and HeLa cells with UV irradiation and radioactive analog BLM, respectively, followed by cell survival analysis (Cell). survival assay) was performed.

Specifically, the cell survival assay was performed as follows: UV and BLM were treated to the control and PIG3 knockdown HCT116 and HeLa cells, respectively, and then 5 × 10 ² cells were inoculated into 60 mm dishes and colonies were obtained. Incubate at 37 ° C. for 2-3 weeks to form. Colonies were counted after staining with 2% methylene blue / 50% ethanol. Fractions of live cells were calculated as the ratio of the plating efficiency of the treated cells to the untreated cells. All data were determined with mean ± standard deviation through three independent experiments, and statistical comparisons were performed using an unpaired t test. p values <0.01 were considered statistically significant.

As a result, PIG3 knockdown cells showed significantly higher sensitivity to UV and BLM compared to control cells (FIGS. 1B and 1C).

Experimental Example 3 Investigation of DNA Damage Recovery in PIG3 Knockdown Cells

In order to determine whether increased sensitivity to DNA damaging factors of PIG3 knockdown cells affects defects in repairing DNA damage, we used UV in PIG3 knockdown cells using pulsed-field gel electrophoresis (PFGE). The extent of repair activity against induced DNA damage was investigated. After UV irradiation to the PIG3 knockdown cells and the control cells, respectively, the degree of DNA double-strand break (DSB) retention was confirmed.

Specifically, the chromosome fingerprinting was performed as follows: To investigate the degree of DSB rejoining, cells were first exposed to 10 J / m 2 UV irradiation. After various recovery times (0, 4, 8, 12, 16 and 20 hours), the cells were resuspended in 1% CleanCut Agarose (Bio-Rad, Hercules, CA) and then placed into an agarose plug. . Solidified agarose plugs were prepared using 1% N-lauroyl sarcosine, 100 mM EDTA [pH 8.0], according to manufacturer's instructions (Roche Diagnostic Corp., Indianapolis, IN). It was dissolved overnight in 0.2% sodium deoxycholate, and 1 mg / ml proteinase K. After washing the plug in washing buffer, CHEF gel electrophoresis was performed using the CHEF DRII instrument (Bio-Rad) at 14 ℃, 0.5 × TBE having a field strength of 1.5 V / cm. Pulse time increased from 50 to 5000 over 50-65 hours. Gels were stained with 0.5 μg / ml EtBr (ethidium bromide). DNA fraction (% DNA extracted) transferred from the plug into the lane was measured using a UV transilluminator and analyzed using Scion image software (Scion Corp., Frederick, MD).

As a result, PIG3 knockdown cells maintained DSB at a higher rate than control cells (FIG. 2). Control HCT116 and HeLa cells efficiently recovered most of the DSB with less than 20% DNA cleavage remaining unbound for 20 hours, whereas PIG3-deficient HCT116 and HeLa cells were left unbound and about 40-50 As a percentage, DSB recovery showed a high defect. Thus, it was found that PIG3 knockdown impaired the ability to repair DNA damage.

Experimental Example 4 Investigation of the Effect of PIG3 Knockdown on Intra-S and G2 / M DNA Damage Checkpoints

We investigated whether PIG3 is involved in the regulation of the Intra-S and G2 / M checkpoints following DNA damage. First, to determine the role of PIG3 in the Intra-S stage checkpoint, the control and PIG3 deficient HeLa cells were treated with UV, BLM and NCS, respectively, and the degree of DNA synthesis inhibition was analyzed by bromodioxyuridine binding assay [BrdU ( bromodeoxyuridine) incorporation assay].

Specifically, the BrdU binding assay was performed as follows: In order to examine the cell population in step S, the binding of BrdU was monitored using the parameters of DNA synthesis as directed by the manufacturer (Roche Diagnostic). Control and PIG3-deficient cells were seeded in 48-well plates and treated with NCS, BLM and UV for 24 hours, respectively. After 24 hours, 10 μM BrdU was added to the culture medium to bind into freshly synthesized DNA and then incubated at 37 ° C. for 2 hours. After fixing the cells, intracellular DNA was partially lysed by nuclease treatment. Peroxidase-labeled antibodies against BrdU and peroxidase substrate were added sequentially to produce colored products at the rate of BrdU levels incorporated into intracellular DNA. The colored product was measured at 405 nm using a microplate reader having a standard wavelength at approximately 490 nm. Relative DNA synthesis was calculated as the ratio of absorbance of cells treated with DNA damaging factor to absorbance of control cells. All data were determined with mean ± standard deviation through three independent experiments, and statistical comparisons were performed using an unpaired t test. p values <0.01 were considered statistically significant.

As a result, Hela control cells treated with UV, BLM and NCS, respectively, showed a significant degree of inhibition of DNA synthesis, whereas PIG3-deficient cells efficiently inhibited intra-S stage checkpoint activity, as shown in FIG. 3A. Low levels of DNA synthesis inhibition were shown (FIG. 3A). Thus, it was found that PIG3 plays an important role in intra-S stage checkpoint activity.

In addition, to investigate the effect of PIG3 knockdown at the G2 / M checkpoint, the inventors also treated the control and PIG3 deficient HeLa cells mock-treated or treated with NCS or UV followed by a G2 / M checkpoint assay ( G2 / M checkpoint analysis) was performed.

Specifically, the G2 / M checkpoint analysis is a method previously reported (Kolas, NK, et al., Science , 318, 1637-1640, 2007; Reinhardt, HC, et al., Cancer Cell , 11, 175- 189, 2007). Control and PIG3 deficient HeLa cells were treated with 200 ng / ml NCS and 10 J / m2 UV, respectively, and then inoculated in 100 ng / ml nocodazole-containing medium for 3 hours. The cells were harvested, washed with PBS and then fixed with 1% formaldehyde at 37 ° C. for 10 minutes. The cells were cooled for 1 minute and then permeated overnight at 90 ° C. with 90% methanol. The fixed cells were washed with PBS and then blocked with culture buffer (PBS with 0.5% BSA) for 10 minutes. The cells were stained with anti-phosphate-histone H3 (S10) -Alexa Fluor 647-binding antibody (Cell Signaling Technology, Boston, MA) diluted 1:10 in culture buffer for 1 hour at room temperature in the dark. , Washed and then resuspended in PBS containing 50 μg / ml propidium iodide (PI). At least 10,000 cells were analyzed by fluorescence-activated cell sorting (FACSort, Becton Dickinson, San Jose, Calif.). Acquired data were analyzed using CellQuest Pro software (Becton Dickinson).

As a result, as shown in FIGS. 3B and 3C, control cells were stopped at the G2 stage, whereas most PIG3 deficient cells progressed mitosis (FIGS. 3B and 3C). Thus, it was found that PIG3 plays an important role in the DNA damage checkpoint involved in resistance to DNA damage.

Experimental Example 5 Effect of PIG3 Knockdown on Chk1 and Chk2 Phosphorylation after DNA Damage

The present inventors investigated whether PIG3 plays a role in regulating the Chk1 / Chk2 signaling pathway in response to NCS in order to determine if the molecular properties of cell cycle defects are observed in PIG3 knockdown cells. After NCS treatment in control and PIG3-deficient HeLa cells, phosphorylation of Chk2 was examined using immunoblotting in the same manner as in <Example 1>.

As a result, as shown in FIG. 4A, the degree of phosphorylation of Chk2 was inhibited in P68-deficient cells, Thr68, which is an active site when treated with NCS compared to control cells (FIG. 4A). Control HeLa cells accumulated Thr68-phosphorylated Chk2 after 3 hours of NCS treatment and persisted for 6 hours after NCS treatment. In contrast, PIG3 deficient cells significantly reduced the phosphorylation of Chk2 at certain time points. Thus, the level of unphosphorylated Chk2 indicates whether the cell expresses a control or PIG3 siRNA, indicating that the lack of Chk2 phosphorylation in PIG3-deficient cells is not due to PIG3 regulating endogenous Chk2 levels.

In addition, since activated Chk2 is involved in phosphorylation and degradation of the kinase Cdc25A (Sancar, A., et al., Annu Rev Biochem , 73, 39-85, 2004), NCS-exposed controls and PIG3- Expression levels of Cdc25A in deficient HeLa cells were examined using immunoblotting in the same manner as in <Example 1>.

As a result, as shown in FIG. 4B, PIG3-deficient HeLa cells did not show a sharp decrease in Cdc25A expression level after NCS treatment compared to control cells (FIG. 4B).

In addition, the effect of PIG3 knockdown on the activity of Chk1 by UV irradiation was investigated in the same manner as above.

As a result, as shown in Figure 4c, PIG3-deficient HeLa cells showed a decrease in the UV response Chk1 Ser345 phosphorylation compared to the control cells. Control HeLa cells accumulated Ser345-phosphorylated Chk1 6 hours after UV irradiation, whereas PIG3 deficient HeLa cells significantly inhibited Chk1 phosphorylation (FIG. 4C).

Therefore, PIG3 was found to play a role in regulating Chk1 / Chk2 in response to DNA damage.

Experimental Example 6 Effect of PIG3 Knockdown on Phosphorylation of H2AX after DNA Damage

We investigated the phosphorylation of histone variant H2AX to determine if cytotoxic effects and checkpoint avoidance resulting from unstable checkpoint signals are observed in PIG3 knockdown cells. To investigate whether foci formation of γ-H2AX appeared in control HeLa and HCT116 cells, and in PIG3 knockdown HeLa and HCT116 cells, immunofluorescence microscopy analysis was used.

Specifically, the immunofluorescence microscopic analysis was performed as follows: To visualize intranuclear aggregates, UV, NCS on cells incubated in a cover slip coated with poly-L-lysine (Sigma) And BLM, respectively, and then recovered for the appropriate time. The cells were washed twice with PBS, then fixed with 4% paraformaldehyde for 10 minutes and frozen 98% methanol for 5 minutes and then permeated with 0.3% Triton X-100 at room temperature. The cover slip was then washed three times with PBS and then blocked in PBS with 5% BSA for 1 hour. The cells were either alone or double immunostained with primary antibodies against various proteins overnight at 4 ° C. The cells were then washed with PBS and stained with Alexa Fluor 488- (green, Molecular Probes) or Alexa Fluor 594- (red, Molecular Probes) binding secondary antibodies. After washing, the cells were fixed using a Vectashield mounting medium containing 4,6 diamidino-2-phenylindole (Vector Laboratories, Burlingame, Calif.). . Fluorescence images were taken using a Zeiss Axioplan 2 imaging fluorescence microscope equipped with a charge-coupled device camera and ISIS software (MetaSystems, Altlussheim, Germany).

At this time, the PIG3 aggregate (foci) was searched by immunofluorescence staining using a PIG3 antibody in a 1:50 dilution. γ-H2AX was searched by IF and WB including mouse monoclonal antibody, clone JBW301 (Upstate Biotechnology, Temecula, Calif.) at 1: 200 and 1: 1000 dilutions, respectively. H2AX antibodies were purchased from Upstate. The following antibodies were used for immunofluorescence staining: anti-53BP polyclonal antibody (Santa cruz, 1:50), anti-ATM protein kinase pS1981 monoclonal antibody (Rockland, Immunochemicals Ins., 1: 500), anti- DNA-PK polyclonal antibody (Santa cruz, 1:50), and anti-ATR polyclonal antibody (Santa cruz, 1:50).

As a result, as shown in FIGS. 5A and 5B, control HeLa and HCT116 cells immediately showed the induction of aggregation formation of γ-H2AX, and the aggregation formation was larger and brighter at about 30 minutes after NCS and UV treatment, respectively. (FIG. 5A and FIG. 5B). γ-H2AX aggregate count decreased 24 hours after treatment with NCS and UV, respectively. On the other hand, PIG3 knockdown cells rapidly formed aggregation of γ-H2AX after NCS and UV treatment compared to control cells, but the aggregation degree of γ-H2AX was significantly reduced. The observed number of aggregates of γ-H2AX did not correspond to DSB. This may be due to weak γ-H2AX signals below detection titers.

In addition, the effect of PIG3 on H2AX phosphorylation after DNA damage was examined using immunoblotting of the same method as in <Example 1>.

As a result, as shown in FIGS. 5C and 5D, PIG3-deficient cells showed significantly reduced H2AX phosphorylation after UV and NCS treatment, which is consistent with the results of immunofluorescence analysis. Control HeLa and HCT116 cells increased rapidly within 1 to 3 hours after exposure to NCS and UV, respectively (FIGS. 5C and 5D), whereas PIG3 knockdown cells showed little phosphorylation of H2AX compared to control cells.

Thus, it can be seen that PIG3 deficiency inhibits the signal of DNA damage to H2AX protein, indicating that PIG3 is involved in signals that cause DNA repair as well as the activity of checkpoints in response to UV or radioactive analogs. Could.

Experimental Example 7 Investigation of the Nucleus Location of the N-terminal Region of PIG3

Although PIG3 is reported to be located in the cytoplasm of H1299 cells expressing p53 (Flatt, PM, et al. Cancer Lett , 156, 63-72, 2000), signaling proteins related to DNA damage response often function in the nucleus.

Therefore, we investigated the intracellular location of PIG3 after DNA damage. First, nuclear and cytosolic fractions were prepared from UV treated or untreated HCT116 cells, and then analyzed for PIG3 expression in each fraction.

Specifically, the preparation of the intracellular fractions was performed as follows: After collecting HCT116 cells, cytoplasmic extract buffer containing a protease inhibitor (Roche) [CEB; 10 mM HEPES [pH 7.5], 3 mM MgCl 2, 14 mM KCl, 5% glycerol, 1 mM DTT] for 10 minutes in the refrigerator. For complete dissolution, vortexing was performed for 10 seconds after addition of 0.2% NP-40. After centrifugation at 8,600 × g for 2 minutes, the supernatant (cytoplasmic extract) was transferred to a new tube. The pellet was washed three times in CEB and then in nuclear extract buffer containing a protease inhibitor (NEB; 10 mM HEPES [pH 7.5], 3 mM MgCl 2, 400 mM NaCl, 5% glycerol, 1 mM DTT). After dissolving at 4 ° C. for 30 minutes, it was centrifuged at 13,200 rpm for 30 minutes. The supernatant contained a nuclear extract. The cytoplasm and nuclear fractions were found to be successful fractionation by confirming that alpha-tubulin and γ-H2AX were incompatible with each fraction.

As a result, PIG3 was located mainly in the cytoplasmic fraction, but about 30% was present in the nucleus (FIG. 6A).

In addition, in order to further confirm the intracellular location of PIG3, a full length (GFP-tagged full-length), C-terminal (1-480 nt) deficient mutation of PIG3 labeled with GFP prepared as in Example 3 above. And transforming the expression constructs encoding the N-terminal (421-999 nt) deficient mutations (Fig. 6B) into HCT116 cells, respectively, and then the level of PIG3 expression from each construct was determined using PIG3 and GFP antibodies. It was analyzed by Western blotting as in <Experimental Example 1>.

As a result, as shown in Fig. 6c, it was confirmed that the N-terminal part (1-480 nt) of PIG3 is an essential part in the nucleus position, and this part is increased by UV irradiation (Fig. 6c). Therefore, PIG3 is located in both nucleus and cytoplasm, and PIG3 in nucleus is involved in DNA damage signal.

Experimental Example 8 PIG3 Forms Intranuclear Aggregation in Response to DNA Damage and Colocalizes with γ-H2AX

The inventors investigated whether PIG3 forms UV- or NCS-induced intranuclear aggregates using immunofluorescence staining in the same manner as in <Experimental Example 6>.

As a result, HeLa and HCT116 cells not treated with UV or NCS showed disperse nuclear staining (Fig. 7a), but cells treated with UV and NCS, respectively, PIG3 immediately formed intranuclear aggregates and the number of aggregates and Degree increased.

In addition, the location of aggregates of DNA damage induced PIG3 was examined to see if PIG3 aggregation induced by UV or NCS represents the actual site of DNA break.

As a result, it was confirmed that the aggregates of PIG3 were co-located with γ-H2AX and 53BP1 aggregates, and there was physical interaction with γ-H2AX (FIGS. 7B to 7D). Thus, it was found that PIG3 aggregation induced by UV or NCS represents a site of DSB processing, and that PIG3 is upstream of the DNA damage signal.

In addition, ATM, ATR and DNA-PK proteins have been reported to be involved in the formation of aggregates of the MRN complex, γH2AX and 53BP1 (Yang, J., et al., Carcinogenesis , 24, 1571-1580, 2003).

We investigated whether PIG3 aggregate formation was dependent on PIKK. The degree of PIG3 aggregate formation was observed in response to DNA damage after treatment with PIKK inhibitor Wortmannin.

As a result, PIG3 aggregates were reduced after wortmannin treatment (FIG. 8A).

In addition, the degree of aggregate formation of PIG3 was observed in HeLa cells deficient by transducing ATM, ATR and DNA-PK with their specific siRNAs.

As a result, there was no significant difference in the degree of PIG3 aggregate formation between control cells and cells knocked down ATM, ATR and DNA-PK, respectively (FIGS. 8B to 8D). Thus, it was found that PIG3 aggregation is caused by one or more of the PIKK families.

In addition, it was examined whether PIG3 aggregate formation was dependent on p53 since PIG3 is a downstream target gene of p53.

As a result, PIG3 aggregates were observed after DNA damage in HCT116 p53 ^{− / −} cells transformed with PIG3 (FIG. 8E). Therefore, PIG3 aggregate formation was found to be dependent on p53.

The following are some examples of formulation methods containing PIG3 of the present invention, but the present invention is not limited thereto.

Production Example 1 Tablet (Direct Pressurization)

5.0 mg of PIG3 was prepared by mixing and pressing 14.1 mg of lactose, 0.8 mg of crospovidone USNF and 0.1 mg of magnesium stearate.

Production Example 2 Tablet (Wet Assembly)

5.0 mg of PIG3 was mixed with 16.0 mg of lactose and 4.0 mg of starch. 0.3 mg of polysorbate 80 was dissolved in pure water and then an appropriate amount of this solution was added and then atomized. After drying, the fine particles were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The granules were pressed to make tablets.

Preparation Example 3 Powder and Capsule

5.0 mg of PIG3 was mixed with 14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone, and 0.2 mg of magnesium stearate. The mixture was prepared using a suitable apparatus. Filled in 5 gelatin capsules.

Production Example 4 Injection

Injections were prepared by containing 100 mg of PIG3, and besides, 180 mg of mannitol, 26 mg of Na ₂ HPO ₄ 12H ₂ O and 2974 mg of distilled water.

Preparation Example 5 Preparation of Cosmetics in Emulsion Formulation

To the cosmetic composition was prepared in the composition shown in Table 1 . The manufacturing method is as follows.

1) The mixture which mixed the raw materials of 1-9 was heated at 65-70 degreeC.

2) 10 was added to the mixture of step 1).

3) The mixture of the raw materials of 11-13 was heated at 65-70 degreeC, and was completely dissolved.

4) While passing through step 3), the mixture of 2) was slowly added and emulsified for 2 to 3 minutes at 8,000 rpm.

5) The raw material of 14 was dissolved in a small amount of water, and then added to the mixture of step 4) and emulsified for 2 minutes.

6) 15 to 17 of the raw materials were weighed, respectively, and added to the mixture of step 5) and emulsified for 30 seconds.

7) After emulsifying the mixture of step 6) through a degassing process to prepare a cosmetic of emulsion type by cooling to 25 ~ 35 ℃.

Composition of Emulsifying Formulations 1, 2, 3 Furtherance Emulsifier 1 Emulsifier 2 Emulsifier 3 One Stearic acid 0.3 0.3 0.3 2 Steali alcohol 0.2 0.2 0.2 3 Glyceryl Monostearate 1.2 1.2 1.2 4 Beeswax 0.4 0.4 0.4 5 Polyoxyethylene sorbitan
Monolauric acid ester 2.2 2.2 2.2 6 Methyl paraoxybenzoate 0.1 0.1 0.1 7 Paraoxybenzoic Acid Profiles 0.05 0.05 0.05 8 Cetylethylhexanoate 5 5 5 9 Triglyceride 2 2 2 10 Cyclomethicone 3 3 3 11 Distilled water To 100 To 100 To 100 12 Concentrated glycerin 5 5 5 13 Triethanolamine 0.15 0.15 0.15 14 Polyacrylic acid polymer 0.12 0.12 0.12 15 Pigment 0.0001 0.0001 0.0001 16 incense 0.10 0.10 0.10 17 PIG3 0.0001 One 10

Preparation Example 6 Preparation of Cosmetics in Solubilized Formulations

To prepare a cosmetic of the solubilized formulation in the composition described in Table 2 . The manufacturing method is as follows.

1) Raw materials 2 to 6 were placed in raw material 1 (purified water) and dissolved using a still mixer.

2) The raw materials of 8 to 11 were put into the raw material of 7 (alcohol) and completely dissolved.

3) The mixture of step 2) was solubilized with slow addition to the mixture of step 1).

Composition of Solubilized Formulations 1, 2, 3 Furtherance  Solubilized Formulation 1 Solubilized Formulation 2  Solubilized Formulation 3 One Purified water  To 100  To 100  To 100 2 Concentrated glycerin  3  3  3 3 1,3-butylene glycol  2  2  2 4 EDTA-2Na  0.01  0.01  0.01 5 Pigment  0.0001  0.0002  0.0002 6 PIG3  0.1 5 5 7 Alcohol (95%)  8 8 8 8 Methyl paraoxybenzoate  0.1  0.1  0.1 9 Polyoxyethylene
Hydrogenated Ester  0.3  0.3  0.3 10 incense  0.15  0.15  0.15 11 Cyclomethicone   -   -  0.2

PIG3 of the present invention can be used to study how to increase cell resistance to DNA damage caused by DNA damage factors such as ultraviolet rays or radioactive drugs, and inhibits the expression of PIG3 in cancer cells, thereby reducing cell resistance to DNA damage. It can be used to study the method of promoting apoptosis by DNA damage factor.

1A is a diagram showing the expression level of PIG3 analyzed using Western blot analysis in PIG3 knockdown HCT116 and HeLa cells including a PIG3 shRNA expression vector.

FIG. 1B is a graph showing cell viability analyzed using Cell survival assay after UV treatment in PIG3 knockdown HCT116 and HeLa cells.

FIG. 1C is a graph showing cell viability analyzed using cell survival analysis after bleomycin (BLM) treatment in PIG3 knockdown HCT116 and HeLa cells.

2 is a graph showing the degree of recovery of DNA double strand breaks (DSBs) analyzed using Pulsed-Field Gel Electrophoresis (PFGE) after UV treatment in PIG3 knockdown HCT116 and HeLa cells. .

Figure 3a shows the degree of DNA synthesis analyzed using UV, BLM and neocarzinostatin (NCS) in PIG3 knockdown HeLa cells, respectively, using bromodioxyuridine incorporation assay [BrdU (bromodeoxyuridine) incorporation assay] A graph representing.

FIG. 3B is a fluorescence-activated cell using G2 / M checkpoint analysis after mock-treated, NCS or UV treatment in PIG3 knockdown HeLa cells. It is a graph showing the number of pH3 positive cells (degree of cells present in the M stage) measured by sorting).

FIG. 3C is a graph showing the relative pH3 positive cells compared to integrate the data of FIG. 3B. FIG.

Figure 4a is a diagram showing the phosphorylation of Chk2 analyzed by Western blot analysis after NCS treatment in PIG3 knockdown HeLa cells.

4B is a diagram showing the expression level of Cdc25A analyzed using Western blot after NCS treatment in PIG3 knockdown HeLa cells.

Figure 4c is a diagram showing the phosphorylation of Chk1 analyzed by Western blot analysis after UV treatment in PIG3 knockdown HeLa cells.

FIG. 5A is a diagram showing the degree of foci formation of γ-H2AX analyzed by immunofluorescence microscopy after NCS treatment in PIG3 knockdown HeLa cells. FIG.

Figure 5b is a diagram showing the degree of foci formation of γ-H2AX analyzed by immunofluorescence microscopy after UV treatment in PIG3 knockdown HeLa cells.

Figure 5c is a diagram showing the expression level of γ-H2AX analyzed by Western blot after NCS treatment in PIG3 knockdown HCT116 and HeLa cells.

5d is a diagram showing the expression level of γ-H2AX analyzed by Western blot analysis after UV treatment in PIG3 knockdown HCT116 and HeLa cells.

Figure 6a is a diagram showing the degree of PIG3 expression analyzed by Western blot analysis after preparing the nuclear and cytoplasmic fractions (fractions) from UV treated or untreated HCT116 cells.

FIG. 6B shows expression constructs encoding GFP-tagged full-length, C-terminal (1-480 nt) deficient mutations, and N-terminal (421-999 nt) deficient mutations of PIG3 labeled with GFP. This is a picture that shows.

FIG. 6C is a diagram showing the expression level of PIG3 analyzed by Western blot analysis after UV treatment in the cells transformed with the HCT116 cells, respectively.

Figure 7a is a diagram showing the degree of nucleus aggregate formation analyzed by immunofluorescence microscopy after treatment with UV and NCS in HeLa cells, respectively.

FIG. 7B is a diagram showing aggregate positions of PIG3 and γ-H2AX analyzed by immunofluorescence microscopy after treatment with UV, BLM and NCS in HeLa cells, respectively.

Figure 7c is a diagram showing the position of PIG3 and 53BP1 aggregates analyzed by immunofluorescence microscopy after treatment with UV, BLM and NCS in HeLa cells, respectively.

Figure 7d is a diagram showing the binding of γ-H2AX and PIG3 analyzed by immunofluorescence microscopy after UV treatment in HeLa cells.

8A is a diagram showing the degree of formation of PIG3 aggregates analyzed by immunofluorescence microscopy after treatment with PIKK inhibitor Wortmannin in HeLa cells.

FIG. 8B shows the depletion of ATM by transformation with ataxia telangiectasia mutated (ATM) siRNA in HeLa cells, followed by UV or NCS treatment, and analysis of PIG3 using immunofluorescence microscopy, respectively. This figure shows the degree of aggregate formation.

FIG. 8C shows the aggregates of PIG3 analyzed by immunofluorescence microscopy after UV or NCS treatment, respectively, after depletion of ATR by transformation with ATM and Rad3 related protein (ATR) siRNA in HeLa cells. This figure shows the degree of formation.

Figure 8d is transformed with DNA dependent protein kinases (DNA-PK) siRNA in HeLa cells, deficient in DNA-PK, treated with UV or NCS, respectively, and analyzed using immunofluorescence microscopy. This figure shows the degree of aggregate formation of PIG3.

Figure 8e is a diagram showing the degree of aggregate formation of PIG3 analyzed by immunofluorescence microscopy after treatment with UV, BLM and NCS in HCT116 p53 ^-/- cells transformed with PIG3, respectively.

<110> Industry-Academic Cooperation Foundation, Chosun University <120> A method for protecting DNA damage using PIG3 <130> 8p-12-45 <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 332 <212> PRT <213> Homo sapiens <400> 1 Met Leu Ala Val His Phe Asp Lys Pro Gly Gly Pro Glu Asn Leu Tyr   1 5 10 15 Val Lys Glu Val Ala Lys Pro Ser Pro Gly Glu Gly Glu Val Leu Leu              20 25 30 Lys Val Ala Ala Ser Ala Leu Asn Arg Ala Asp Leu Met Gln Arg Gln          35 40 45 Gly Gln Tyr Asp Pro Pro Pro Gly Ala Ser Asn Ile Leu Gly Leu Glu      50 55 60 Ala Ser Gly His Val Ala Glu Leu Gly Pro Gly Cys Gln Gly His Trp  65 70 75 80 Lys Ile Gly Asp Thr Ala Met Ala Leu Leu Pro Gly Gly Gly Gln Ala                  85 90 95 Gln Tyr Val Thr Val Pro Glu Gly Leu Leu Met Pro Ile Pro Glu Gly             100 105 110 Leu Thr Leu Thr Gln Ala Ala Ala Ile Pro Glu Ala Trp Leu Thr Ala         115 120 125 Phe Gln Leu Leu His Leu Val Gly Asn Val Gln Ala Gly Asp Tyr Val     130 135 140 Leu Ile His Ala Gly Leu Ser Gly Val Gly 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sapiens <400> 2 ccagccgtcc attccggtgg aggcagaggc agtcctgggg ctctggggct cgggctttgt 60 caccgggacc cgcaggagcc agaaccactc ggcgccgcct ggtgcatggg aggggagccg 120 ggccaggagt aagtaactca tacgggcgcc ggggacccgg gtcgggctgg gggcttccaa 180 ctcagaggga gtgtgatttg cctgatcctc ttcggcgttg tcctgctctg ccgcatccag 240 ccctgtaccg ccatcccact tcccgccgtt cccatctgtg ttccgggtgg gatcggtctg 300 gaggcggccg aggacttccc aggcaggagc tcggggcgga ggccgggtcc gcggcagacc 360 agggcagcga ggcgctggcc ggcagggggc gctgcggtgc cagcctgagg ctgggctgct 420 ccgcgaggat acagcggccc ctgccctgtc ctgtcctgcc ctgccctgtc ctgtcctgcc 480 ctgccctgcc ctgtcctgtc ctgccctgcc ctgccctgtg tcctcagaca atatgttagc 540 cgtgcacttt gacaagccgg gaggaccgga aaacctctac gtgaaggagg tggccaagcc 600 gagcccgggg gagggtgaag tcctcctgaa ggtggcggcc agcgccctga accgggcgga 660 cttaatgcag agacaaggcc agtatgaccc acctccagga gccagcaaca ttttgggact 720 tgaggcatct ggacatgtgg cagagctggg gcctggctgc cagggacact ggaagatcgg 780 ggacacagcc atggctctgc tccccggtgg gggccaggct cagtacgtca ctgtccccga 840 agggctcctc atgcctatcc cagagggatt gaccctgacc caggctgcag ccatcccaga 900 ggcctggctc accgccttcc agctgttaca tcttgtggga aatgttcagg ctggagacta 960 tgtgctaatc catgcaggac tgagtggtgt gggcacagct gctatccaac tcacccggat 1020 ggctggagct attcctctgg tcacagctgg ctcccagaag aagcttcaaa tggcagaaaa 1080 gcttggagca gctgctggat tcaattacaa aaaagaggat ttctctgaag caacgctgaa 1140 attcaccaaa ggtgctggag ttaatcttat tctagactgc ataggcggat cctactggga 1200 gaagaacgtc aactgcctgg ctcttgatgg tcgatgggtt ctctatggtc tgatgggagg 1260 aggtgacatc aatgggcccc tgttttcaaa gctacttttt aagcgaggaa gtctgatcac 1320 cagtttgctg aggtctaggg acaataagta caagcaaatg ctggtgaatg ctttcacgga 1380 gcaaattctg cctcacttct ccacggaggg cccccaacgt ctgctgccgg ttctggacag 1440 aatctaccca gtgaccgaaa tccaggaggc ccataagtac atggaggcca acaagaacat 1500 aggcaagatc gtcctggaac tgccccagtg aaggaggatg gggcaggaca ggacgcggcc 1560 accccaggcc tttccagagc aaacctggag aagattcaca atagacaggc caagaaaccc 1620 ggtgcttcct ccagagccgt ttaaagctga tatgaggaaa taaagagtga actgg 1675 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> sense RNA <400> 3 aaauguucag gcuggagacu a 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> antisense RNA <400> 4 uagucuccag ccugaacauu u 21 <210> 5 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA duplex <400> 5 ccuacgccaa uuucgu 16 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA duplex <400> 6 acgaaauugg uggcguagg 19 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PIG3 sense primer <400> 7 aaggaaataa ccaccatgtt agccgtgcac 30 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> P223 antisense primer <400> 8 ctggggcagt tccaggacga tctt 24 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> C-terminal deletion PIG3 sense primer <400> 9 aaggaaataa ccaccatgtt agccgtgcac 30 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> C-terminal deletion PIG3 antisense primer <400> 10 gagttggata gcagctgtgc ccaca 25 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> N-terminal deletion PIG3 sense primer <400> 11 aaggaaaraa ccaccatggg agactatgtg cta 33 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> N-terminal deletion PIG3 antisense primer <400> 12 ctggggcagt tccaggacga tctt 24  

Claims (17)

A cancer treatment aid containing an inhibitor of the expression or activity of PIG3 (p53-inducible gene 3). The adjuvant of claim 1, wherein the PIG3 has an amino acid sequence set forth in SEQ ID NO: 1. The method of claim 1, wherein the inhibitor of expression of PIG3 is antisense nucleotide, small interfering RNA (siRNA), or small hairpin RNA (shRNA) that complementarily binds to mRNA of PIG3 gene. Cancer treatment supplements. According to claim 1, wherein the activity inhibitor of PIG3 is any one selected from the group consisting of compounds, peptides, peptide mimetics and antibodies that bind to PIG3 complementary to cancer therapy. The cancer treatment aid according to claim 1, wherein the cancer is any one selected from the group consisting of rectal cancer, colon cancer, colon cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, and liver cancer. A method for preventing or treating cancer, comprising administering an inhibitor of PIG3 expression or activity to cancer cells. The method of claim 6, wherein the PIG3 has an amino acid sequence set forth in SEQ ID NO: 1. The method of claim 6, wherein the inhibitor of expression of PIG3 is antisense nucleotide, small interfering RNA (siRNA) or small hairpin RNA (shRNA) that complementarily binds to the mRNA of the PIG3 gene. Cancer prevention or treatment. 7. The method of claim 6, wherein the inhibitor of PIG3 activity is any one selected from the group consisting of compounds, peptides, peptide mimetics and antibodies that bind to PIG3 complementarily. The method of claim 6, wherein the cancer is any one selected from the group consisting of rectal cancer, colon cancer, colon cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, and liver cancer. Anti-aging cosmetic composition containing PIG3 (p53-inducible gene 3) as an active ingredient. The anti-aging cosmetic composition according to claim 11, wherein the PIG3 has an amino acid sequence represented by SEQ ID NO: 1. Anti - aging cosmetic composition containing PIG3 ( p53-inducible gene 3 ) gene construct as an active ingredient. The anti-aging cosmetic composition according to claim 13, wherein the PIG3 gene has a nucleotide sequence set forth in SEQ ID NO: 2. Cell resistance to DNA damaging agents, comprising administering to the cell a composition comprising PIG3 (p53-inducible gene 3) or PIG3 ( p53-inducible gene 3 ) gene construct as an active ingredient How to increase. The method of claim 15, wherein the DNA damaging factor is ultraviolet (UV) or radiomimetic drug. 17. The method of claim 16, wherein the radioactive drug is bleomycin or neocarzinostatin.

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