KR100898866B1 - A composition and a method for inducing apotosis of cancer cell using dek overexpression and a drosophila melanogaster overexpressing dek - Google Patents

Abstract

The present invention relates to a composition for inducing apoptosis, comprising a DEK protein or a nucleic acid encoding the DEK protein, and a method for inducing apoptosis by overexpressing DEK. The present invention also provides a transformed Drosophila that overexpresses DEK protein.

Description

COMPOSITION AND A METHOD FOR INDUCING APOTOSIS OF CANCER CELL USING DEK OVEREXPRESSION AND A DROSOPHILA MELANOGASTER OVEREXPRESSING DEK}

The present invention relates to a composition for inducing apoptosis, comprising a DEK protein or a nucleic acid encoding the DEK protein, and a method for inducing apoptosis by overexpressing a DEK protein. The present invention also provides a transformed Drosophila overexpressing human DEK protein and a method for producing the same.

DEK, also known as the original oncogene protein, is a protein associated with acute myeloid blood tumors caused by 6 and 9 translocations on the chromosome by altering the charge of the DNA, causing positive supercoils to cause DNA phase changes in the chromatin. For example, in acute myeloid leukemia, DEK is found in the form of DEK-CAN protein by fusion with CAN protein, and mRNA of DEK is increased in liver cancer, malignant glioma and melanoma. In addition, DEK proteins containing acidic domains are involved in transcriptional regulation by binding to histones, inhibiting the acetylation of H3 and H4 and inhibiting the activity of p300 and PCAF, known as histone acetylases (Ko SI, Lee). IS, Kim JY, Kim SM, Kim DW, Lee KS, Woo KM, Baek JH, Choo JK, Seo SB. "Regulation of histone acetyltransferase activity of p300 and PCAF by proto-oncogene protein DEK", FEBS LETTERS 580, 3217 ~ 3222 (2006, May 29).

In addition, DEK binds to p65, a subunit of NF-κB, and regulates autoimmune-related diseases and cancers, where DEK inhibits the activity of NF-κB and the expression of NF-κB related genes, and NF-κB Inhibition of the expression of the NF-κB reporter gene, Mcp-1 and IkBa gene by binding to the promoter region of the clAP2 and IL-8 gene under the control of. Increased binding of P / CAF, an activator of the promoter region of these genes, in cells with reduced DEK expression has confirmed that DEK is associated with intracellular transcription inhibition (Sammons M, Wan SS, Vogel). NL, Mientjes EJ, Grosveld G, Ashburner BP. "Negative regulation of the RelA / p65 transactivation function by the product of the DEK proto-oncogene", J. Biol. Chem. 281, 26802-26812 (2006, Sep. 15) ] Reference).

As described above, the type of cancer associated with DEK is increasing, but the role of DEK is still unknown. In addition, there is no current report on the association between the role of histone proteins in the regulation of transcription of cellular genes and the role of apoptosis.

Recently, a variety of intracellular roles of proteins including acidic domains have been suggested. In particular, subunits of INHAT (inhibitor of histone acetyltransferase), pp32 and SET / TAF-Ibeta, have been reported to be involved in the regulation of apoptosis.

Acidic protein pp32 promotes the formation of apoptosome, a complex of protease, thereby promoting the activity of Caspase-9 and Caspase-3, leading to caspase dependent apoptosis. In particular, the acidic protein prothymosin-alpha, purified with the INHAT complex, has also been found to play an important role in the apoptosis pathway (Jiang X, Kim HE, Shu H, Zhao Y, Zhang H). , Kofron J, Donnelly J, Burns D, Ng SC, Rosenberg S, Wang X. "Distinctive roles of PHAP proteins and prothymosin-alpha in a death regulatory pathway", Science 299, 223-226 (2003, Jan. 10)] Reference).

Another acidic protein of the INHAT complex, SET / TAF-Ibeta, is a cell produced by caspase-dependent pathways in which granzyme A secreted from cytotoxic T lymphocytes causes cleavage of single-stranded DNA by DNAases. When killing, it has been reported to play an important role in the cell death pathway by inhibiting the DNAase and exhibiting inhibitory activity of apoptosis (Fan Z, Beresford PJ, Oh DY, Zhang D, Lieberman J. "Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its inhibitor", Cell 112, 659-672 (2003 Mar. 7)].

In the present invention, DEK protein acts on the promoter region of bcl-2 in addition to the promoter region of the previously reported genes to inhibit the acetylation of histones H3 and H4, thereby inhibiting the expression of cytochrome C in mitochondria. By inducing release to induce the activity of caspase-9 and caspase-3, the function of inducing apoptosis in cells was further elucidated.

In addition, in the present invention, by producing an anticancer substance using a protein present in the living body, the anticancer substance according to the present invention can achieve the minimization of side effects that are difficult to expect from chemicals, and effectively play a role in the living body more effectively anticancer Can provide an effect.

Furthermore, in the present invention, a transformed Drosophila overexpressing DEK protein can be provided. The transformed Drosophila overexpressing the DEK protein of the present invention can confirm the apoptosis inducing effect of the DEK protein according to the present invention as well as provide a model for studying the function of the DEK protein.

Document 1 Documents such as Ko SI, Lee IS, Kim JY, Kim SM, Kim DW, Lee KS, Woo KM, Baek JH, Choo JK, Seo SB. "Regulation of histone acetyltransferase activity of p300 and PCAF by proto-oncogene protein DEK", FEBS LETTERS 580, 3217-3222 (2006, May 29)]

Document 2 Sammons M, Wan SS, Vogel NL, Mientjes EJ, Grosveld G, Ashburner BP. "Negative regulation of the RelA / p65 transactivation function by the product of the DEK proto-oncogene", J. Biol. Chem. 281, 26802-26812 (2006, Sep. 15)]

Document 3Jiang X, Kim HE, Shu H, Zhao Y, Zhang H, Kofron J, Donnelly J, Burns D, Ng SC, Rosenberg S, Wang X. "Distinctive roles of PHAP proteins and prothymosin-alpha in a death regulatory pathway ", Science 299, 223-226 (2003, Jan. 10)]

Document 4 Fan Z, Beresford PJ, Oh DY, Zhang D, Lieberman J. "Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its inhibitor" , Cell 112, 659-672 (2003 Mar. 7)]

It is therefore an object of the present invention to provide compositions and methods for inducing apoptosis, in particular for apoptosis of cancer cells.

Still another object of the present invention is to provide a transformed Drosophila induced by apoptosis and a method of preparing the same.

In order to achieve the above object, the present invention provides a composition and method for overexpressing DEK protein to induce apoptosis of cancer cells.

In order to achieve the above another object, the present invention provides a Drosophila transformed to overexpress DEK protein.

In accordance with the present invention, by overexpressing DEK in the cell, the caspase dependent apoptosis is increased, thereby exhibiting an anticancer effect of inhibiting abnormal growth of cells, that is, cancer cells, and thus can be usefully used for the prevention and treatment of various cancers.

In the present invention, DEK protein inhibits the expression of bcl-2 by inhibiting the acetylation of histones H3 and H4 in the promoter region of bcl-2, an apoptosis inhibiting gene, thereby inducing the release of cytochrome C from mitochondria. It was newly discovered that induction of apoptosis by inducing the activity of caspase-9 and caspase-3, which are death inducing proteins.

As such, the DEK protein may inhibit aberrant proliferation of cells, resulting in overexpression of DEK, which may have an effect of inhibiting tumor by causing cell death.

In one embodiment, the present invention relates to a composition for inducing apoptosis, comprising as an active ingredient a DEK protein or a nucleic acid encoding a DEK protein.

The DEK protein is characterized by being represented by the amino acid sequence of SEQ ID NO: 2, but not limited to 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably the amino acid sequence. May comprise a protein represented by an amino acid sequence having at least 95% homology.

In addition, the nucleic acid encoding the DEK protein is characterized by being represented by the base sequence of SEQ ID NO: 1 or a base sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more. .

DEK protein in the present invention can be obtained by chemical synthesis or using genetic recombination techniques. When prepared by chemical synthesis, it can be obtained using a polypeptide synthesis method well known in the art. In the case of genetic recombination technology, a nucleic acid encoding a DEK protein is inserted into an appropriate expression vector, the vector is transformed into a host cell, the host cell is cultured so that the DEK protein is expressed, and then the DEK protein is recovered from the host cell. Can be obtained. Proteins are expressed in selected host cells and then subjected to conventional biochemical separation techniques, such as treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography for isolation and purification. (Gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like can be used. In general, these are used in combination to separate proteins of high purity.

In addition, when used as a gene therapy agent, the DEK coding gene of the present invention can be administered directly by injection. Alternatively, a vector comprising the DEK coding gene of the present invention may be administered, and specific examples of suitable vectors include adenovirus vectors, adeno-associated vectors, herpes virus vectors, vaccinia virus vectors, and retroviral vectors.

Apoptosis inducing agent having a DEK protein or a nucleic acid encoding the same as an active ingredient, and a pharmaceutical composition for treating apoptosis-related diseases may be administered in a suitable formulation with a carrier and diluent known in the art, And parenteral administration, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppositories and the like. When the therapeutic agent of the present invention is administered orally, the therapeutic agent may be administered in the form of tablets, capsules, particles, powders, pills, troches, solutions, suspensions, emulsifiers, or syrups.

The formulations are suitable excipients, fillers, binders, wetting agents, disintegrating agents, lubricants, surfactants, dispersants, buffers, preservatives, dissolution aids, disinfectants, sweeteners, spices, analgesics, stabilizers, isotonic agents commonly used in pharmaceutical compositions. It may be produced by a conventional method using the same.

Each formulation described above may comprise a pharmaceutically acceptable carrier or additive. Specific examples of the carrier or additive include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidine, carboxyvinyl polymer, sodium alginate, water soluble dextran, carboxymethyl sodium starch, pectin, xanthan rubber , Gum arabic, casein, gelatin, agar, glycerol, propylene glycol, polyethylglycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactic acid. One or more additives may be selected or appropriately combined depending on the type of preparation. Furthermore, as a method of administering a gene therapy treatment, topical administration to target cells may be performed in addition to conventional systemic administration such as intravenous and intraarterial administration, and a combination of catheter technique and surgical operation may be used. have.

At this time, the cancer of the composition of the present invention may exhibit a therapeutic effect, specifically, breast cancer, ovarian cancer, liver cancer, lung cancer, colon cancer, prostate cancer and the like, but is not limited thereto.

The pharmaceutical composition of the present invention comprises a DEK protein or nucleic acid encoding the same, in a pharmaceutically effective amount, together with a pharmaceutically acceptable carrier.

In the present invention, the "pharmaceutically effective amount" refers to the amount of the active ingredient that exhibits an inhibitory, ameliorating and / or cure effect against the cancer to be treated. The dosage of the DEK protein of the present invention or the nucleic acid encoding the same varies depending on the weight, age, sex, health condition, diet, time of administration, administration method and severity of disease of the patient. For example, therapeutically effective dosages can be initially determined using in vitro assays through cell culture, and performed in animal models to determine the IC50 (in vitro) concentration range of blood DEK protein determined in cell culture. In assays, drug treatments, the concentration of the test compound at a lethal dose relative to 50% of the cultured cells) can be determined. It will be possible to determine the amount effective for treatment without undue experimentation in the art, and this information can be used to more accurately determine the useful dose in humans.

The present invention also provides a Drosophila transformed with an expression vector containing a gene encoding a DEK protein.

The transgenic Drosophila overexpressing the DEK protein may be prepared by introducing a recombinant expression vector expressing the DEK protein into the fertilized egg to induce overexpression of the DEK. Specifically, preparing a DEK expression vector; Micro-injecting the expression vector into the nucleus of the embryo; It provides a transgenic fruit flies comprising the method of culturing the embryo.

The transgenic Drosophila can be obtained by injecting a DEK protein expression vector into a Drosophila embryo within 1 hour after spawning and culturing the embryo with the DEK. Specifically, in the present invention, a microinjection method of directly injecting the vector into the microcapillary tube and directly injecting the embryo removed into the egg film may be used.

The transgenic Drosophila prepared by the above method not only confirms the apoptosis effect of DEK, but can also be used as a model for exploring further functions of DEK protein in the future.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention should not be construed as being limited by these examples.

Example  1: in cancer cells DEK Of cell death by overexpression

An expression vector for overexpressing DEK protein in cancer cells was prepared and transfected into the cells, followed by reverse transcriptase-polymer chain reaction (RT-PCR) and Western blot. After confirming the overexpression of DEK in the cell death was measured by FACS (Fluorescence Activated Cell Sorting Analysis) analysis. Specific experiments were carried out as follows, and the results are shown in FIG.

1-1 DEK  Preparation of Expression Vector

PCMX PL1 and pcDNA3.1 expression vectors capable of expressing DEK in cells were used to determine whether apoptosis was induced upon overexpression of DEK in cancer cells. Cells were prepared in a HeLa cell line (Manassas, ATCC, VA) in a 35 mm dish to 2.5 × 10 ⁵ cell numbers, incubated for 48 hours at 37 ° C., 5% CO ₂ , and trizol from these cells. (Invitrogen) solution was used to extract the entire RNA using the manufacturer's recommendations, and then synthesized cDNA using M-MLV reverse transcriptase (Promega) as the template as a template It was. To amplify the nucleic acid encoding the DEK gene, insertion into the pCMX PL1 vector is reversed with a forward primer (5'-CGC GGA TCC GCG ATG TCC GCC TCG GCC CCT GCT GCG-3 ', SEQ ID NO: 3) synthetically for the nucleic acid. A primer (5'-CGC GAA TTC GCG TCA AGA AAT TAG CTC TTT TAC-3 ', SEQ ID NO: 4) was used, and a forward primer (5'-CGC GGA TCC GCG ATG for nucleic acid for insertion into pcDNA3.1 vector) Polymerase chain reaction (PCR) using TCC GCC TCG GCC CCT GCT GCG-3 ', SEQ ID NO: 5) and reverse primer (5'-CGC GAT ATC GCG TCA AGA AAT TAG CTC TTT TAC-3', SEQ ID NO: 6) , Polymerase Chain Reaction), and the PCR was initially denatured at 94 ° C. for 5 minutes and then repeated 30 times at 94 ° C. for 30 seconds, 61 ° C. for 1 minute, and 72 ° C. for 1 minute 30 seconds. BamH I and EcoR I (pCMX PL1) and BamH I and EcoR V (pcDNA3.1) Using restriction enzymes, DEK expression vectors were prepared by specifically cleaving both ends of the PCR product and the expression vector and inserting them into the expression vector.

1-2 Cell Culture and Transfection

HeLa cell line (Manassas, Inc., VA) was prepared using DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum (FBS, fetal bovine serum, Invitrogen) and penicillin-streptomycin (50 units / ml). Incubated for 12 hours. The cells were washed with DMEM medium containing no FBS and transfected with HeLa cells by injecting expression vectors of pCMX PL1-DEK and pcDNA3.1-DEK using Lipofectamine (Invitrogen). I was. The control group was injected into Hela cells using pCMX PL1 and pcDNA3.1 empty vectors without gene insertion. Thereafter, the transfected cells were incubated for 72 hours at 37 ° C. and 5% CO ₂ .

1-3 DEK Cell death caused by overexpression

FACS analysis was performed to confirm the induction of apoptosis upon overexpression of DEK in cancer cells. Cell lysis buffer (1% triton X-100, 150mM NaCl, 50mM Tris-HCl (pH 8.0), 0.1% SDS, 1% NP-40, 1mM PMSF, 1X protease inhibitor cocktail) After lysing, the cell lysate was centrifuged at 12,000 rpm for 10 minutes to obtain a supernatant of protein. This supernatant was transferred to a new tube. In order to confirm the overexpression of DEK in the transfected cells, proteins of each sample were separated by electrophoresis through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic protein was transferred to a nitrocellulose membrane, and blotting was performed using an anti-DEK antibody. Secondary antibodies were prepared using anti-mouse-IgG HRP conjugated with Horse Radish Peroxidase, and developed on X-ray films using a chemiluminescence substrate (Chemiluminescence, Santa Cruz). DEK overexpression was detected. Also, in order to confirm the amount of mRNA of DEK, a primer (5'-CGC GGA TCC GCG ATG TCC GCC TCG) capable of specifically amplifying DEK after extracting total RNA through the method described in Example 1-1 above was used. GCC CCT GCT GCG-3 ', SEQ ID NO: 7) and reverse primers (5'-CGC GAA TTC GCG TCA AGA AAT TAG CTC TTT TAC-3', SEQ ID NO: 8) confirmed the increase in the amount of DEK mRNA . It was performed in the same manner using the beta-actin gene as a control thereof. As a result, as shown in Figure 1a, it was confirmed that the DEK gene overexpressed about 2 to 3 times in the transfected cells than the non-transfected cells (see Figure 1a).

In order to confirm the increase of apoptosis due to overexpression of DEK, the transfected cells were isolated in a dish using trypsin-EDTA, followed by centrifugation at 2,000 rpm for 5 minutes. Cells precipitated through centrifugation were washed with phosphate buffered saline (PBS) and Annexin-V (annexin) using Annexin-V-fluoride kit (Roche Applied Science) as recommended by the manufacturer. -V) and propidium iodide (PI) were co-labeled on the cells. Subsequently, labeling of 10,000 cells was recorded and the increase in apoptosis in control and DEK overexpressing cells was analyzed using FACS. As a result of analysis of apoptosis, as shown in FIG. 1B, when a cell transfected with pCMX PL1-DEK was compared with a cell treated with nothing, it was confirmed that apoptosis was increased by 15% in cells transfected with pCMX PL1-DEK. . In addition, it was confirmed that apoptosis was increased by 9.2% when comparing the transfected cells with the pCMX-PL1 covector (see FIG. 1B). Therefore, it was confirmed that cell death can be increased by overexpressing DEK in cancer cells.

Example  2: DEK  Overexpressed cells Caspase -9 and Caspase -3 increases activity

In order to determine whether overexpression of DEK protein induces apoptosis through increased activity of proteolytic enzymes, the activation of caspase-9 and 3, a potent proteolytic enzyme among apoptosis effectors in the apoptosis pathway, was investigated. It was confirmed in DEK overexpressing cells. In addition, the amount of active caspase-9 protein and caspase-9 proprotein in DEK overexpressing cells was confirmed by Western blot. Specific experiments were performed as follows, and the results are shown in FIG. 2.

2-1 Caspase -9 and Caspase -3 activity measurement

HeLa cell lines were prepared to be 2.5 × 10 ⁵ cell numbers in a 35 mm dish, incubated for 24 hours at 37 ° C., 5% CO ₂ , and then transfected with pCMX PL1-DEK or pcDNA3.1-DEK. The transfected HeLa cells were disrupted with the cell lysates of Examples 1-3, and the cell lysates were centrifuged at 12,000 rpm for 10 minutes to obtain supernatants of proteins. This supernatant was transferred to a new tube. Caspase-9 using Caspase-Glo 9 assay kit (Promega) and Apopprobe-3 assay kit (Peptron) as recommended by the manufacturer And 3 activity was measured. The activity of caspase-9 is fluorinated using LEHD (L: Leucine, E: Glutamic acid, H: Histidine, D: Aspartic acid) conjugated with proluminesence as a substrate for caspase-9. The degree of activity was quantified using FLUOstar Optima (BMG Labtech). The activity of caspase-3 is 360 nm excitation wavelength and 460 nm based on DEVD (D: Aspartic acid, E: Glutamic, V: Valine) -AMC conjugated to 7-amino-4-methylcoumarin (AMC) The degree of activity was measured using a microplate fluorescent reader Victor 3 (Perkin Elmer) at the emission wavelength of.

2-2 Weston Blotting

Proteins were isolated via SDS-PAGE to confirm an increase in the caspase-9 in active form and a decrease in the proprotein of caspase-9. This was carried out using a Western blot using an anti-caspase-9 proprotein antibody or an anti-active caspase-9 antibody in the same manner as described in Examples 1-3, in the active form of caspase-9 and caspase- 9 proproteins were detected.

 As a result, as shown in Figure 2, it was confirmed that the caspase-9 activity was increased 5.5-fold in HeLa cells transformed with pCMX PL1-DEK or pcDNA3.1-DEK compared to the cells treated with nothing. In addition, it was confirmed that the caspase-3 activity was increased four-fold in HeLa cells transformed with pCMX PL1-DEK or pcDNA3.1-DEK compared to the cells treated with nothing (see FIG. 2A).

In addition, the detection of active caspase by Western blotting reduced the amount of 46Kd caspase-9 proprotein in cells transfected with pCMX PL1-DEK, and the 35Kd active caspase-9 protein. It was confirmed that this increased (see Fig. 2b). From this, when DEK is overexpressed intracellularly, the proproteins of caspase-3 and caspase-9 are cleaved and turned into active form, and their activity is increased, so that the proteins of these active forms are potent against other proteins. It was confirmed that it acts as a degrading enzyme to induce apoptosis.

Example  3: DEK On by bcl -2 regulates expression regulation and inhibition

DEK protein is known as a regulator that inhibits gene transcription by inhibiting acetylation of histones by having inhibitory activity of histone acetyltransferase (HAT). Therefore, in order to confirm that apoptosis is induced by negatively regulating transcription of a gene that inhibits apoptosis, chromatin immunoprecipitation is used at a promoter region that regulates transcription of a representative apoptosis inhibitory gene, bcl-2. The degree of acetylation of histone was confirmed. In addition, when DEK was overexpressed, quantitative RT-PCR was performed to confirm the expression level of mRNA of bcl-2 in the cells, and the amount of bcl-2 protein was confirmed by Western blotting. Specific experiments were performed as follows, and the results are shown in FIG. 3.

3-1 Chromatin  Immunoprecipitation Chromatin Immunoprecipitation )

HeLa cell lines were cultured in 100 mm dishes and transfected with pCMX-DEK. Incubated for 72 hours after transfection, these cells were crosslinked with each other protein and DNA for 10 minutes at 37 ℃ using 1% formaldehyde (formaldehyde). Then, 10-fold glycine was treated at room temperature for 5 minutes to stop the crosslinking reaction. These cells were transferred to SDS lysis buffer using a cell scraper and short cuts of DNA cross-linked with the protein via an ultrasonic mill. Subsequently, the supernatant was transferred to a new tube by centrifugation, and then, anti-acetyl histone H4 antibody and anti-acetyl histone H3 antibody were added to the cell lysate and reacted at 4 ° C. for 20 hours. In order to perform immunoprecipitation, protein A / G agarose beads were added and reacted for 1 hour. Then, the antibody-associated protein was precipitated, and tris-HCl and EDTA were added to the DNA in the precipitate where the protein and DNA were bound to each other. Was extracted. Using the extracted DNA as a template, a specific forward primer (5'-CCA GGC AGC TTA ATA CAT TCT TTT TAG-3 ', SEQ ID NO: 9) and a reverse primer (5'-TGA TGC) for the promoter region of bcl-2 were used. PCR was performed using TGA AAG GTT AAA GAA AAA AC-3 ′, SEQ ID NO: 10).

3-2 Quantitative Reverse Transcriptase Polymerase Chain Reaction

HeLa cell lines were cultured in 35 mm dishes and transfected with pCMX-DEK. 72 hours after transfection, cDNA was synthesized from these cells using the Trizol (Invitrogen) solution in the same manner as described in Example 1-1, and the promoter region of the bcl-2 gene was synthesized. Quantitative reverse transcriptase polymerase chain reaction was performed using a combination of primers to amplify. Thereafter, the ABI prism 7900 sequence detection system (ABI prism 7900 detection system, Applied Biosystems, Inc.) was analyzed.

3-3 Weston Blotting

HeLa cell lines were cultured in 35 mm dishes, transfected with pCMX PL1-DEK, and cultured for 72 hours. The transfected HeLa cells were disrupted with cell lysate and centrifuged at 12,000 rpm for 10 minutes to obtain supernatant of protein in cell lysate. After transferring the supernatant thereof to a new tube, proteins were separated by SDS-PAGE to measure the amount of bcl-2 protein expression in the transfected cells, and the difference in expression level was detected using anti-bcl-2 antibody. . Anti-beta-actin antibody was used as a control.

As a result, as shown in FIG. 3, when chromatin immunoprecipitation using the anti-acetyl histone H3 antibody and the anti-acetyl histone H4 antibody was performed, the histone of the promoter region of the bcl-2 gene in cells overexpressing DEK It was confirmed that acetylation of H3 and histone H4 was reduced (see FIG. 3A). In addition, when the quantitative reverse transcriptase polymerase chain reaction was performed, it was confirmed that the mRNA expression level of the bcl-2 gene in the DEK overexpressing cells decreased sharply (see FIG. 3B), and the amount of bcl-2 protein was decreased through Western blot. (See FIG. 3C).

These results confirm that DEK inhibits the transcriptional activity of bcl-2 by inhibiting the acetylation of histone H3 and histone H4 at the promoter region of the bcl-2 gene when overexpressed in cells. bcl-2 has a function of inhibiting apoptosis by inhibiting the release of cytochrome C from the mitochondria, while DEK releases cytochrome C by inhibiting the function of bcl-2, resulting in caspase-9 activity. It was confirmed that inducing apoptosis by subsequently increasing the activity of caspase-3.

Example  4: DEK Production of Transgenic Drosophila Overexpressing

Transgenic Drosophila was established by microinjection with P transfer factor (Spradling and Rubin, 1982). Specific experiments were performed as follows.

4-1 Construction of vector for Drosophila transformants

 The HeLa cell line was incubated in a 35 mm dish, using Trizol (Invitrogen) solution as recommended by the manufacturer, extracting total RNA from these cells, and using this as a template, the M-MLV reverse transcriptase (Promega) was prepared. CDNA was synthesized using as recommended. Specific forward primers ('5-ATG TCC GCC TCG GCC CCT GC-3', SEQ ID NO: 11) and reverse primers ('5-TCA AGA) with sequences cleaved with EagI and XbaI restriction enzymes capable of amplifying the DEK gene PCR was performed using AAT TAG CTC TTT ACA-3 ′, SEQ ID NO: 12) and inserted into the pGEMT vector by TA cloning. Recombinant vector pUAS-hDEK was prepared by inserting the 1128 bp DEK coding sequence of pGEMT-DEK prepared by the above method into a pUAST vector digested with the same restriction enzyme by cleavage with EagI and XbaI restriction enzymes (see FIG. 4A). ).

4-2 human DEK Fruit flies that can overexpress Transitional  making

Embryos of Drosophila Melanogaster within 1 hour after spawning from Bloomington stock center, Indiana University to produce transgenic Drosophila were collected using vinegar-dry yeast plates. The eggs were removed using 2.6% sodium hypochlorite solution and arranged on a slide with double-sided tape. The arranged embryos were identified under an Olympus reversed-phase microscope, and DNA for transformation was directly injected into the embryo ends using an Olympus microinjection manipulation system (IMT-2). The recombinant vector pUAS-hDEK and the helper vector (Δ2-3) were mixed and injected into microcapillary tubes prepared from Narishige's PB-7 capillary (0.7 mm inner diameter) extractor to remove the egg yolk. Fine injection. Apply halocarbon oil 700 (Sigma) to micro-injected embryos and incubate in a 25 ° C incubator for 24 hours.The first stage larvae that have emerged from embryos are screened and transferred to the standard medium of fruit flies. Incubate until The adult fruit flies were again crossed with the white-eye fruit flies, and transformants were selected for the fruit flies with the red-eye phenotype.

Example  5: human DEK Analysis of Apoptosis Using Drosophila Transformants Overexpressing

Expression of human DEK mRNA and protein in overexpressed lines by reverse transcriptase polymerase chain reaction and Western blotting using human DEK overexpressing transformants to confirm the function of human DEK expected to induce apoptosis in Drosophila Was measured. In addition, it was confirmed that apoptosis was induced in overexpressed lines through phenotypic analysis and caspase-3 activity measurement. Specific experiments were performed as follows, and the results are shown in FIG. 5.

5-1 human DEK Establishment of system overexpressing

Overexpression of DEK was performed using the UAS-Gal4 system (Brand and Perrimon, 1993). Induction of overexpression of human DEK was achieved by establishing a GMR> hDEK line by crossing with a GMR-Gal4 line capable of specifically inducing expression in the fruit of pUAS-hDEK and Drosophila eyes.

5-2 Reverse Transcriptase Polymerase Chain Reaction RT - PCR )

Compared with wild-type (WT), human DEK inserted but not expressed (UAS-hDEK) and UAS-hDEK strains and induction strains (GMR-Gal4) capable of expressing human DEK in Drosophila eyes when crossed with In order to confirm whether human DEK mRNA was overexpressed in the human DEK overexpression line (GMR> hDEK) that crossed the UAS-hDEK and GMR-Gal4 lines, the adult of each line was selected and subjected to reverse transcriptase polymerase chain reaction. After treatment with trizol to the selected adult, and pulverized with a cell crusher to extract the total RNA, using this as a template to synthesize cDNA using M-MLV reverse transcriptase (Promega), specific forward direction specific for DEK gene PCR was amplified using a primer ('5-ATG TCC GCC TCG GCC CCT GC-3', SEQ ID NO: 13) and a reverse primer ('5-TCA AGA AAT TAG CTC TTT ACA-3', SEQ ID NO: 14). As a control for the expression level of DEK, rp49 gene was used (see FIG. 5A).

5-3 Weston  Blotting ( Western blotting )

In order to confirm the overexpression of human DEK protein in human DEK overexpressing strains compared to each control group used in Example 5-2, adults of each strain were selected, and treated with cell lysate, and then pulverized with a cell grinder and centrifuged. After that, the proteins of each strain were extracted. The extracted protein was isolated through SDS-PAGE, and overexpression of DEK was detected in the overexpressed line using an anti-DEK antibody. Anti-beta-actin was used as a control (see FIG. 5A).

5-4 human DEK  Phenotypic Analysis Associated with Apoptosis in Overexpressed Systems

To determine whether overexpression of DEK in a line that overexpresses human DEK causes apoptosis in the eye of Drosophila, controls (WT), UAS-hDEK, GMR-Gal4 and GMR> hDEK + inhibitor and overexpression lines (GMR phenotypes appearing in the eyes of> hDEK) were analyzed. In the control group, GMR> hDEK + inhibitor is a line inducing an inhibitory response to caspase 3 in human DEK overexpression line (GMR> hDEK). After anesthetizing adult strains, the phenotypes of the eyes of the normal control group and the eyes of the overexpressing transformants were observed with a scanning electron microscope (Leo 1455VP Environmental Scanning Electron Microscope, LEO) (see FIG. 5B).

5-5 Transformants Caspase -3 increases activity

In order to measure the caspase-3 activity of the transformants overexpressing human DEK compared to the respective control lines, the adult of each line was treated with a cell lysate, and then pulverized with a cell crusher and centrifuged. Apopprobe-3 assay kit (Peptron) was used in the above-described method using the extracted protein of each strain, and GMR> hDEK + inhibitor of the control group was used for the activity of caspase-3. Treatment of caspase-3-substrate with cell lysates extracted with DEVD (D: Aspartic acid, E: Glutamic acid, V: Valine) conjugated to aldehydes (CHO, aldehyde) from GMR> hDEK control Inhibition of caspase-3 was induced by adding 0.1 mM before 5 minutes below, and the level of activity was quantified using Victor 3 (Perkin Elmer) (see FIG. 5C).

As shown in FIG. 5B, the phenotype of abnormal cell differentiation in which the shape and size of the single eye forming the compound eye of the regular arrangement of the fruit flies in the transformant overexpressing the human DEK when compared to the eyes of the normal control group was destroyed. It was confirmed (see FIG. 5B). Thus, it can be seen that abnormal cell differentiation is caused by the induction of cell death by overexpression of DEK. In addition, as a result of measuring caspase-3 activity used as a representative marker of apoptosis in overexpressing transformants, caspase-3 overexpression of human DEK was observed in Drosophila transformants compared to normal control in Drosophila transformants. It was confirmed that the activity is increased by 99% (see FIG. 5C).

Therefore, it was confirmed that apoptosis is induced in the transformant overexpressing human DEK, and this phenomenon was confirmed to be induced by caspase dependence in the apoptosis pathway.

Figure 1a is a result of overexpressing the result of overexpression after transfection of DEK of the present invention into cells through a reverse transcriptase polymerase chain reaction.

1B is a graph confirming the result of apoptosis after transfection of DEK of the present invention into cells by Western blotting.

Figure 2a is a graph confirming the increase of caspase-9 and caspase-3 activity in the cells overexpressed DEK protein of the present invention by Western blotting.

Figure 2b is the result of detecting the expression of caspase-9 of the active form in the cell overexpressed DEK protein of the present invention.

Figure 3a is a result showing that histone acetylation of the promoter region of the bcl-2 gene was inhibited by the DEK protein of the present invention.

Figure 3b is a graph detecting that the expression level of mRNA of the bcl-2 gene is reduced by the DEK protein of the present invention.

Figure 3c is the result of detecting that the expression level of the protein of the bcl-2 gene is reduced by the DEK protein of the present invention.

Figure 4 shows the cloning of cDNA of human DEK to the Drosophila transformation vector pUAST using Eagl and Xbal restriction enzyme.

Figure 5a is a result confirmed by the reverse transcriptase and Western blotting in the transformant overexpressing human DEK.

5B shows the results of observing phenotypes of the eyes of normal controls and the eyes of transformed Drosophila overexpressing human DEK.

5C shows the results of apoptosis induction and caspase-3 activity in the eyes of transformants overexpressing human DEK.

<110> Seoul National University Industry Foundation          Chung Ang University Industry-Academic Cooperation Foundation <120> A composition and a method for inducing apotosis of cancer cell          using DEK overexpression and a drosophila melanogaster          overexpressing DEK <130> FPD / 200709-0040 <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 1128 <212> DNA <213> Homo sapiens <400> 1 atgtccgcct cggcccctgc tgcggagggg gagggaaccc ccacccagcc cgcgtccgag 60 aaagaacccg aaatgcccgg tcccagagag gagagcgagg aggaagagga cgaggacgac 120 gaggaggagg aggaggagga aaaagaaaag agtctcatcg tggaaggcaa gagggaaaag 180 aaaaaagtag agaggttgac aatgcaagtc tcttccttac agagagagcc atttacaatt 240 gcacaaggaa aggggcagaa actttgtgaa attgagagga tacatttttt tctaagtaag 300 aagaaaaccg atgaacttag aaatctacac aaactgcttt acaacaggcc aggcactgtg 360 tcctcattaa agaagaatgt gggtcagttc agtggctttc catttgaaaa aggaagtgtc 420 caatataaaa agaaggaaga aatgttgaaa aaatttagaa atgccatgtt aaagagcatc 480 tgtgaggttc ttgatttgga gagatcaggt gtaaatagtg aactagtgaa gaggatcttg 540 aatttcttaa tgcatccaaa gccttctggc aaaccattgc cgaaatctaa aaaaacttgt 600 agcaaaggca gtaaaaagga acggaacagt tctggaatgg caaggaaggc taagcgaacc 660 aaatgtcctg aaattctgtc agatgaatct agtagtgatg aagatgaaaa gaaaaacaag 720 gaagagtctt cagatgatga agataaagaa agtgaagagg agccaccaaa aaagacagcc 780 aaaagagaaa aacctaaaca gaaagctact tctaaaagta aaaaatctgt gaaaagtgcc 840 aatgttaaga aagcagatag cagcaccacc aagaagaatc aaaacagttc caaaaaagaa 900 agtgagtctg aggatagttc agatgatgaa cctttaatta aaaagttgaa gaaaccccct 960 acagatgaag agttaaagga aacaataaag aaattactgg ccagtgctaa cttggaagaa 1020 gtcacaatga aacagatttg caaaaaggtc tatgaaaatt atcctactta tgatttaact 1080 gaaagaaaag atttcataaa aacaactgta aaagagctaa tttcttga 1128 <210> 2 <211> 375 <212> PRT <213> Homo sapiens <400> 2 Met Ser Ala Ser Ala Pro Ala Ala Glu Gly Glu Gly Thr Pro Thr Gln   1 5 10 15 Pro Ala Ser Glu Lys Glu Pro Glu Met Pro Gly Pro Arg Glu Glu Ser              20 25 30 Glu Glu Glu Glu Asp Glu Asp Asp Glu Glu Glu Glu Glu Glu Glu Lys          35 40 45 Glu Lys Ser Leu Ile Val Glu Gly Lys Arg Glu Lys Lys Lys Val Glu      50 55 60 Arg Leu Thr Met Gln Val Ser Ser Leu Gln Arg Glu Pro Phe Thr Ile  65 70 75 80 Ala Gln Gly Lys Gly Gln Lys Leu Cys Glu Ile Glu Arg Ile His Phe                  85 90 95 Phe Leu Ser Lys Lys Lys Thr Asp Glu Leu Arg Asn Leu His Lys Leu             100 105 110 Leu Tyr Asn Arg Pro Gly Thr Val Ser Ser Leu Lys Lys Asn Val Gly         115 120 125 Gln Phe Ser Gly Phe Pro Phe Glu Lys Gly Ser Val Gln Tyr Lys Lys     130 135 140 Lys Glu Glu Met Leu Lys Lys Phe Arg Asn Ala Met Leu Lys Ser Ile 145 150 155 160 Cys Glu Val Leu Asp Leu Glu Arg Ser Gly Val Asn Ser Glu Leu Val                 165 170 175 Lys Arg Ile Leu Asn Phe Leu Met His Pro Lys Pro Ser Gly Lys Pro             180 185 190 Leu Pro Lys Ser Lys Lys Thr Cys Ser Lys Gly Ser Lys Lys Glu Arg         195 200 205 Asn Ser Ser Gly Met Ala Arg Lys Ala Lys Arg Thr Lys Cys Pro Glu     210 215 220 Ile Leu Ser Asp Glu Ser Ser Ser Asp Glu Asp Glu Lys Lys Asn Lys 225 230 235 240 Glu Glu Ser Ser Asp Asp Glu Asp Lys Glu Ser Glu Glu Glu Pro Pro                 245 250 255 Lys Lys Thr Ala Lys Arg Glu Lys Pro Lys Gln Lys Ala Thr Ser Lys             260 265 270 Ser Lys Lys Ser Val Lys Ser Ala Asn Val Lys Lys Ala Asp Ser Ser         275 280 285 Thr Thr Lys Lys Asn Gln Asn Ser Ser Lys Lys Glu Ser Glu Ser Glu     290 295 300 Asp Ser Ser Asp Asp Glu Pro Leu Ile Lys Lys Leu Lys Lys Pro Pro 305 310 315 320 Thr Asp Glu Glu Leu Lys Glu Thr Ile Lys Lys Leu Leu Ala Ser Ala                 325 330 335 Asn Leu Glu Glu Val Thr Met Lys Gln Ile Cys Lys Lys Val Tyr Glu             340 345 350 Asn Tyr Pro Thr Tyr Asp Leu Thr Glu Arg Lys Asp Phe Ile Lys Thr         355 360 365 Thr Val Lys Glu Leu Ile Ser     370 375 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> forward primer for DEK <400> 3 cgcggatccg cgatgtccgc ctcggcccct gctgcg 36 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for DEK <400> 4 cgcgaattcg cgtcaagaaa ttagctcttt tac 33 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> forward primer for DEK <400> 5 cgcggatccg cgatgtccgc ctcggcccct gctgcg 36 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for DEK <400> 6 cgcgatatcg cgtcaagaaa ttagctcttt tac 33 <210> 7 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> forward primer for DEK <400> 7 cgcggatccg cgatgtccgc ctcggcccct gctgcg 36 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for DEK <400> 8 cgcgaattcg cgtcaagaaa ttagctcttt tac 33 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer for DEK <400> 9 ccaggcagct taatacattc tttttag 27 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for DEK <400> 10 tgatgctgaa aggttaaaga aaaaac 26 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for DEK <400> 11 atgtccgcct cggcccctgc 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for DEK <400> 12 tcaagaaatt agctctttac a 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for DEK <400> 13 atgtccgcct cggcccctgc 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for DEK <400> 14 tcaagaaatt agctctttac a 21  

Claims (8)

delete A composition for treating cancer, comprising a DEK protein represented by the amino acid sequence of SEQ ID NO: 2, or a nucleic acid encoding the DEK protein. delete The method of claim 2, The nucleic acid is a composition characterized in that represented by the nucleotide sequence of SEQ ID NO: 1. delete A method of causing apoptosis by overexpressing a DEK protein represented by the amino acid sequence of SEQ ID NO: 2 in vitro. delete delete

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