KR20090040809A - Method of screening apoptosis inducing anticancer reagents using rhob promoter reporter system - Google Patents

Abstract

A method of screening apoptosis-inducing anticancer reagents by using a RhoB promoter reporter system is provided to fine drug which inhibits the growth of tumor cell and induces the apoptosis, therefore, a therapeutic agent can be developed. A screening system of an improvement material of RhoB gene expression comprises (i) nucleotide encoding a RhoB promoter; and (ii) host cell which contains nucleotide encoding luciferase or cyanogen fluorescence protein(CFP) and is transformed to the plasmid which does not encode the other promoter or enhancers.

Description

Method of Screening Apoptosis Inducing Anticancer Reagents Using RhoB promoter Reporter System}

The present invention relates to a system for measuring the promoter activity of a RhoB protein that induces apoptosis by using a reporter and a method for screening apoptosis inducing substances therethrough.

Cancer is characterized by "uncontrolled growth and proliferation of cells", and the growth and proliferation of these abnormal cells leads to the formation of cell masses called tumors that infiltrate surrounding tissues and, in severe cases, other organs of the body. It may also transition to. On the other hand, the cell mass produced by the growth and proliferation of such abnormal cells is histologically called neoplasia.

There are various causes of cancer, but it is largely divided into internal factors and external factors. Internal factors include genetic factors, immunological factors, and external factors include chemicals, radiation, and viruses. The mechanism by which normal cells are transformed into cancer cells is not known, but at least 80-90% are known to be influenced by external factors such as environmental factors. Genetic factors, one of the internal factors, include oncogenes and tumor suppressor genes, which are genes related to cancer, and when the balance between them is broken down by internal or external factors described above, This will occur.

Cancer is largely classified into blood cancer and solid cancer according to the site of its occurrence, and occurs in almost all parts of the body such as lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer and skin cancer. Among the methods for treating these solid cancers, chemotherapy agents other than surgery or radiation therapy are collectively called anticancer agents, and most of them are substances that exhibit anticancer activity by inhibiting nucleic acid synthesis of cells.

Chemotherapeutic agents are broadly classified into antimetabolites, alkylating agents, antimitotic drugs, and hormones. Among these, metabolic antagonists inhibit the metabolic processes necessary for the proliferation of cancer cells, and include folic acid derivatives such as methotrexate, purine derivatives such as 6-mercaptopurine and 6-thioguanine. Pyrimidine derivatives such as 5-fluorouracil and cytarabine. Alkylating agent is an anti-cancer effect by modifying the structure of the DNA and cutting the chain by introducing an alkyl group to the guanine of DNA, such as chlorambucil (cycloambucil) and cyclophosphamide (cyclophosphamide), nitrogen mustard compounds, thio Ethyleneimine compounds such as thiotepa, alkylsulfonate compounds such as busulfan, nitrosourea compounds such as carmustine, and triazene compounds such as dacarbazine. have. Mitosis inhibitors are mitosis specific drugs that inhibit mitosis and inhibit cell division, including anticancer drugs such as actinomycin D, doxorubicin, bleomycin, and mitomycin; Plant alkaloids such as vincristine and vinblastine; Taxoids, such as mitosis inhibitors including taxane rings, are included. In addition, hormones such as corticosteroids and progesterone and platinum-containing compounds such as cisplatin are used as anticancer agents.

The biggest problem with chemotherapeutic agents is drug resistance, which is the main factor that eventually causes treatment to fail despite the initial successful response by anticancer drugs. In addition to researching the cause of drug resistance, the development of anti-cancer drugs with new mechanisms is needed to treat cancers resistant to existing drugs. Current anticancer drugs include drug resistance blockers, angiogenesis inhibitors, tumor metastasis inhibitors, and drugs targeting gene expression.

Accordingly, the present inventors have been interested in a reporter system that can observe an increase in expression of the RhoB gene that induces apoptosis while studying a method for screening a new mechanism of anticancer agent that suppresses tumor growth and induces apoptosis.

small GTPase RhoB belongs to the Rho family RhoA, RhoB, RhoC, which has been reported to be involved in various intracellular processes such as cytoskeletal composition, gene transcription, cell cycle progression, and cytokines. On the other hand, RhoA and RhoB have 86% amino acids homology, but their functions are completely different. In addition, most Rho proteins act to activate proliferation and transformation into malignant cells, but RhoB may have a function of inhibiting cancer formation and progression. Rho proteins are farnesylated by farnesyltransferse (FTase 1) at the C-terminal CAAX position by isoprenoid lipids or by geranylgeranyltransferase I, Geranylated by GGTase I), responsible for different functions. RhoB is induced by stress such as platelet driven growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor β (TGFβ) and DNA damage. And apoptosis occurs when the expression level is increased, so it is also called "apoptosis inducer".

Increased RhoB expression is directly related to apoptosis by inducing DNA damage and is also affected by the regulation of transcriptional function of NFκB. Increased RhoB expression results in tumor survival, transformation of malignant transformation, and migration by tumorigenic genes such as Epidermal Growth Factor Receptor (EGFR), Ras, and Akt. Apoptosis is induced by inhibiting the functions associated with,, invasion and the like. Farnesyltransferse inhibitors (FTIs) have been reported to increase RhoB expression by inducing DNA damage by reactive oxygen species (ROS). The mechanism of increased RhoB expression is to inhibit the binding of histone deacetylase 1 (HDAC 1) to the position containing CCAAT in the RhoB promoter sequence, thereby activating the acetylated RhoB promoter to increase the amount of RhoB transcription. Reported. Thus, histone deacetylase 1 inhibitors are also known to increase RhoB expression and induce apoptosis.

Therefore, in the present invention, by treating a histone deacetylase 1 inhibitor to increase the expression of RhoB that causes apoptosis, by producing a screening system to confirm this through the reporter system co-transfected with the RhoB gene, RhoB The present invention was completed by confirming that the promoter can be easily screened for an anticancer agent capable of inducing apoptosis by increasing RhoB expression.

It is an object of the present invention to provide a RhoB promoter activity identification system capable of confirming RhoB promoter activity through Drosophila luciferase or Drosophila cyan fluorescent protein (Cyan Fluoresecence Protien, CFP) reporter.

Still another object of the present invention is to provide a method for screening a substance inducing RhoB promoter activity through the luciferase or cyan fluorescent protein reporter system.

In one aspect of the invention, a system for identifying RhoB promoter activity using the Drosophila luciferase reporter system is provided.

Specifically, the RhoB promoter activity confirmation system has no other promoter and enhancer to measure activity of the RhoB promoter alone, and RhoB promoter activity formed by inserting the RhoB gene into a plasmid vector containing a luciferase gene to construct a reporter system. The plasmid vector for confirmation is prepared by introducing into a cancer cell line.

In a preferred embodiment of the present invention, a primer capable of isolating human genomic DNA from a MCF7 cell line (ATCC Cat. No. # HTB-22), which is a breast cancer cell line, and amplifying the RhoB promoter region as a template (base sequence 2). And 3) was used to conduct a polymerase reaction (PCR). The RhoB promoter DNA obtained by the above polymerase reaction has no promoter and enhancer at all, and is inserted into pGL2-basic vector (Promega), which is a plasmid vector capable of expressing luciferase, and the luciferase is produced only according to the activity of the RhoB promoter. The expressed plasmid vector (pGL2-basic-RhoB promoter) was prepared (FIG. 2). The plasmid vector pGL2-basic-RhoB promoter thus prepared is a plasmid vector pLa-SV40 (Promega) containing lanilla luciferase to standardize the fluorescence intensity, and HeLa cell line (ATCC Cat.No. CCL2). The Lanilla luciferase containing vector is used as a standard fluorescent material when treating candidates expected to activate the RhoB promoter in the cell line into which the plasmid vector is introduced and measuring the intensity of luciferase after a certain time.

In a preferred embodiment of the present invention, by using the luciferase reporter system described above, Westeroylanilide hydroxamic acid (Hystone diacetylase 1, HDAC 1) is known to increase the activity of the RhoB promoter ( suberoylanilide hydroxamic acid (SAHA), farnesyltransferase inhibitor-277 (FTI-277), and geranylgeranyltransferase I inhibitor -286, GGTI-286) as positive controls The intensity of luciferase was measured after treatment with 9 NCI drugs. Information on the NCI drugs used can be found on the website (http://dtp.nci.nih.gov/).

Specifically, the HeLa cell line into which the plasmid was introduced was stabilized for 12 hours, and the cells were cultured by adding each of the three positive control substances or 9 NCI drugs to the culture medium for 12 hours. After 12 hours, the cells were lysed with the cell lysis solution, and the fluorescence intensity of luciferase was measured in the cell lysate using a luminometer. The measured fluorescence intensity of luciferase was quantified by normalizing with the intensity of fluorescence of co-introduced lanilla luciferase.

In another aspect of the invention, a system for identifying RhoB promoter activity using a cyan fluorescent protein reporter system is provided.

Specifically, the system substitutes the RhoB promoter in the pTagCFP-N vector, a plasmid vector expressing Cyan Fluorescence Protein (CFP), with the same RhoB promoter as the one inserted into the pGL2-basic-RhoB promoter. To prepare a pTagCFP-N-RhoB promoter vector, a plasmid vector capable of recognizing the activation of fluorescence due to the expression of cyan fluorescent protein (FIG. 4). The pTagCFP-N-RhoB promoter vector thus prepared is prepared by introducing into a HeLa cell line (ATCC Cat. No. CCL2) as in the luciferase reporter system.

In a preferred embodiment of the present invention, using the cyan fluorescent protein reporter system described above, subseroylanilide hydroxamic acid, which is known to increase the activity of the RhoB promoter by inhibiting histone diacetylase 1 (HDAC 1) (suberoylanilide hydroxamic acid, SAHA), and farnesyltransferase inhibitor-277 (FTI-277) as a positive control, Sorafenib irrelevant to inhibition of histone deacetylase 1 Was treated with nine NCI drugs as described above as a negative control, and the intensity of luciferase was measured.

Specifically, the HeLa cell line into which the plasmid was introduced was cultured in a medium containing G418 to selectively culture only the cells containing the vector, and then, when cyan fluorescent colonies were formed, only those portions were taken to stably culture the vector. Was established. After culturing the selected cell line as described above in a culture plate and stabilizing for 12 hours, two positive controls, one negative control, and nine NCI substances were added to the culture medium in the same manner as the Luciferase reporter system described above. Was incubated. After 12 hours, the degree of fluorescence was observed using a fluorescence microscope in the wavelength region where cyan fluorescent protein could be observed. As a result, as shown in FIG. 5, SAHA and FTI-277, which were positive controls, fluoresced at a concentration of 10 uM. The degree of RhoB promoter activity was increased by fluorescence of NSC126188 was brighter than those treated with sorafenib, a negative control among NCI drugs.

In still another aspect of the present invention, there is provided a method for confirming whether or not a substance induces apoptosis of a substance identified by the above screening system to increase the activity of a RhoB promoter.

The occurrence of apoptosis can be confirmed by various methods. The simplest method is a method of confirming DNA fragmentation, which is a characteristic of apoptosis, by electrophoresis, and a TUNEL assay (terminal dUTP nick-end labeling assay) or Anexin V. Flow cytometry using the method is generally used. In particular, Anexin V has a property of binding to phosphatidylserine (PS), which is present in a large amount in the cell membrane at the initial stage of apoptosis, and can be quickly and easily quantitatively analyzed by using a flow cytometer after fluorescent labeling of Anexin V. So it is a widely used method.

In a preferred embodiment of the present invention, Anexin V was used to confirm the occurrence of apoptosis. First, the HeLa cell line was incubated for 18 hours by adding a substance confirmed the RhoB promoter activity using the vector, and then reacted with FITC-labeled Anexin V and analyzed by flow cytometry. As a result, as shown in FIG. 6, more cells were found in the cell group to which the dead cells and the cells combined with the propidium iodide (PI), in which the RhoB promoter activity was confirmed, were added to the substance (bottom center of FIG. 6). ), Cells bound with Annexin V, which bind cells with advanced apoptosis, were also observed more in the cell group to which the material was added (lower right in FIG. 6).

The RhoB promoter active screening system according to the present invention can be used for high-throughput screening (HTS) in drug development.

The fast processing search method is a search method that can obtain superior results in terms of quality and quantity compared to the conventional search method by miniaturizing an existing search unit or automating repetitive tasks. The search can be divided into binding affinity search for purified protein receptor, enzyme activity search, or cell-based function search. Therefore, the RhoB promoter activity screening system according to the present invention can be applied to high speed processing screening methods such as cell arrays.

The RhoB promoter activity screening system of the present invention can easily search for drugs that inhibit the growth of tumor cells and induce apoptosis, and can be used for the development of therapeutic agents for cancer diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples. This invention is not limited by the Example mentioned later, The effect by this invention is not limited by what was described in the Example of this invention.

Example  One. Rhob  Promoter Cloning  And Luciferase  Reporter system building

The RhoB promoter is characterized by genomic DNA isolated from MCF7 cells (ATCC Cat. No. # HTB-22), a breast cancer cell line, using a primer having a sequence of SEQ ID NO: 2 and SEQ ID NO: 3 to perform a polymerase reaction (PCR). As a result, DNA of the RhoB promoter portion (595 bp) corresponding to SEQ ID NO: 1 was obtained.

As a vector for constructing a reporter system, pGL2-basic vector (Promega Co., Ltd.) was used. The vector is suitable for identifying only the activity of the RhoB promoter of the present invention because it is characterized by no promoter and enhancer. First, the RhoB promoter was inserted upstream of the Drosophila luciferase gene in the vector. The RhoB promoter of SEQ ID NO: 1 was designed to include a KpnI restriction enzyme cleavage site so as to be cloned into the pGL2-basic vector. SEQ ID NO: 2 includes a HindIII restriction enzyme cleavage site. In order to insert the RhoB promoter DNA obtained by the polymerase reaction into the pGL2-basic vector, first, the pGL2-basic vector was treated with Kpn I and Hind III for 3 hours, followed by CIP reaction for 30 minutes to remove the phosphate group in the DNA fragment 5'-. It was. In addition, the RhoB promoter DNA obtained by the above polymerase reaction was also ligated with T4 DNA ligase for 16 hours at 16 ° C. with the vector prepared after cutting into Kpn I and Hind III, and the ligation resulted in E. coli competent cells (E. coli competent cells) were introduced into DH5α cells.

After culturing DH5α into which the ligation result was introduced, plasmid DNA was isolated from the grown colonies and cut with Kpn I and HindIII restriction enzymes to confirm the DNA fragment size (595b), and the DNA base was reconfirmed to confirm that the RhoB promoter was cloned. DNA sequencing was performed.

The schematic diagram of the pGL2-RhoB promoter vector including the RhoB promoter and Drosophila luciferase gene finally prepared and sequenced through the above process is shown in FIG. 2.

Example  2. Luciferase  By activity analysis Rhob  Promoter activation compare

After introducing the pGL2-RhoB promoter vector prepared in Example 1 into the cells, the material to be screened was treated to measure the activity of the RhoB promoter by the material.

The prepared pGL2-RhoB- Drosophila luciferase plasmid vector and the plasmid vector containing lanilla-luciferase, pRL-SV40 (Promega), are simultaneously introduced into the HeLa cell line (ATCC Cat. No. CCL2). Cells were stabilized for 12 hours after introduction and treated at concentrations of 5 uM and 10 uM for each drug for 12 hours. As a comparative drug predicted to increase RhoB promoter activity, the analyzed drugs were 9 NCI drugs to measure RhoB promoter activity using HDAC1 inhibitors SAHA, FTI-277, and GGTI-286 as positive controls. NCI drugs used in the experiment are as follows. 1, NSC156272 (C21H19NO6.HCl); 2, NSC126188 (C22H45N2O.I); 3, NSC608781 (C28H36O3); 4, NSC205357 (C5H8N4Se); 5, NSC19703; 6, NSC 67586 (C7H16N2OS2); 7, NSC 233064 (C25H37Cl2N2O3P); 8, NSC287407 (C38H36O6); 9, NSC297364 (C19H17ClN2O). Specific information on the NCI drugs used can be found at the website (http://dtp.nci.nih.gov/).

Drug-treated HeLa cells were treated and washed with cell lysis solution (40 mM Tricine pH 7.8, 50 mM NaCl, 2 mM EDTA, 1 mM MgSO 4, 5 mM DTT, 1% Triton X-100). Light intensity by Drosophila luciferase and Lanilla luciferase activity was synthesized by measuring for 10 seconds with a luminometer (luminometer, Berthold, Bad Wildbad, Germany). Drosophila luciparase activity was analyzed quantitatively by standardizing the ranilla activity value.

As a result, as shown in Figure 3, SAHA, FTI-277, GGTI-286 all increased the intensity of fluorescence more than five times than the cells (CTL on the X-axis in the figure), which was not treated at all at 10 μM concentration, NCI drug In the case of NSC126188 drug (2 on the X-axis) was increased 5.5 times when treated at a concentration of 10 μM. In other drugs, the fluorescence intensity was 1 to 2 times higher than cells treated with no drug, indicating that the RhoB promoter was not activated by the drug.

Example  3. Fluorescent Reporter Gene CFP Screening System Construction

A vector was prepared in which the activity of the RhoB promoter was known through the expression of cyan fluorescence.

The pTagCFP-N vector (Evrogen, Cat. No. FP112) is an expression vector having a TagCFP protein exhibiting cyan fluorescent color. The CMV promoter was removed from the vector and replaced with the RhoB promoter, thereby preparing a plasmid vector capable of recognizing the change in brightness of fluorescence according to RhoB promoter activation.

First, as in Example 1, a genomic DNA isolated from MCF7 cells (ATCC Cat. No. # HTB-22), which is a breast cancer cell line, was used as a template, using a primer having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5. Reaction (PCR) was carried out. As a result, DNA of the RhoB promoter portion (595bp) corresponding to SEQ ID NO: 1 was obtained. At this time, the base sequence 6 was designed to include an Ase I restriction enzyme cleavage site and the base sequence 7 to include a HindIII restriction enzyme cleavage site for cloning the pTagCFP-N vector. In order to convert the unique CMV promoter of the pTagCFP-N vector into the RhoB promoter, the Ase I and HindIII restriction sites were cut for 3 hours in front of and behind the CMV promoter, followed by CIP treatment for 30 minutes. The RhoB promoter DNA obtained by the above enzyme polymerization reaction was also cut with Ase I and HindIII restriction enzymes, and then ligated with T4 DNA ligation for 16 hours at 16 ° C. like the prepared vector, and the ligation resulted in E. coli competent cells (E coli competent cells) were introduced into DH5α cells. Plasmid DNA was extracted from colonies introduced into DH5α, cut with Ase I and HindIII restriction enzymes, and DNA fragment size was confirmed (595 bp). In addition, DNA sequencing confirmed the correct sequencing.

The schematic diagram of the pTagCFP-N-RhoB promoter vector including the RhoB promoter and TagCFP expressed by the above process is shown in FIG. 4.

Example  4. Through cyan fluorescence analysis Rhob  Promoter Activity Measurement

After introducing the pTagCFP-N-RhoB promoter vector prepared in Example 3 into the cells, the material to be screened was treated to measure the activity of the RhoB promoter by the material.

The medium was removed from the HeLa cell line grown to 70% on the surface of 100 mm cell culture dish, and the cell surface was washed with medium without serum and 5 ml of the same medium was added. 6 ug of pTagCFP-N1-RhoB promoter plasmid vector was introduced into HeLa cells using lipofectamine (Invtrogen, Cat. No. 11668-027). When the cells are stabilized, G418 (Sigma, Cat. No. A1720) is added to the medium at 400 μg / ml to select only the cells into which the vector is introduced. When cyan fluorescent colonies were formed on a fluorescence microscope every 24 hours, only those portions were removed and cultured to establish a stabilized cell line expressing cyan fluorescent protein. The cell line was placed in a 30 mm confocal dish and stabilized for 12 hours, and then treated with 12 μh for each of the drugs added to the medium at concentrations of 5 uM and 10 uM. After 12 hours, the degree of fluorescence was observed with a fluorescence microscope equipped with a filter capable of observing TagCFP of Excitation 458 nm and Emission 480 nm. Drugs treated with cell lines were SAHA, a drug that increases RhoB promoter activity, and FTI-277 as a positive control, and Sorafenib (Sorafenib), which was not related to RhoB promoter activity, was used as a negative control. The intensity of cyan fluorescence of nine NCI drugs to be measured for activity was measured.

As a result, SAHA, and FTI-277, the positive controls, increased the fluorescence brightness at 10 μM. In the case of NCI drug, the fluorescence brightness of NSC126188 was greatly increased and in the case of the negative control Sorafenib, there was no change in fluorescence brightness (FIG. 5).

Example  5. NSC126188 On by Rhob  Increase in activity and cellular Apoptosis  Confirm

Based on the results of Examples 2 and 4, it was confirmed whether apoptosis was induced with respect to NSC126188, which was found to increase RhoB activity in the nine NCI drugs tested.

HeLa cells were treated with 10 uM of NSC 126188, reacted with annexin V after 18 hours of incubation, and confirmed by flow cytometry. Annexin V binds to the phosphatidylserine (PS) present in the cell membrane. Phosphatidylserine is present in the cell membrane in the early stage of apoptosis. Thus, the progress of apoptosis can be seen by measuring the amount of phosphatidylserine using annexin V labeled with FITC fluorescence. In this example, the amount of dead cells was also measured by treating with PI (Propidium Iodide) binding to dead cells.

As a result, when NSC126188 was treated, the amount of dead cells increased (lower center of FIG. 6) compared to the cells of the negative control group without treatment (lower center of FIG. 6), and the number of cells binding to annexin was also significantly increased. Was observed (bottom right of FIG. 6). Therefore, it can be seen from these results that the cells treated with NSC126188 caused RhoB promoter activity and apoptosis progressed by the RhoB promoter activity.

  In addition, in order to confirm the increase in the amount of RhoB protein in the cell by RhoB promoter activity, Western blot was performed using the cells after material treatment. First, HeLa cells were treated with NSC126188 (3 μM) and harvested after 4, 6, 8, 10, and 13 hours, respectively, and then RhoB, and proteins related to apoptosis, caspase3, and PARP protein. Western blot was performed to determine if apoptosis was induced.

As a result, it was confirmed that the amount of RhoB protein was rapidly increased after 4 hours of drug treatment. In addition, the amount of activated state in which the cutphase 3, which shows apoptosis, was increased, and the protein of the uncut form of PARP, known as the substrate of caspase 3, was drastically reduced (FIG. 7). As a result, it was confirmed that the drug NSC126188, which activates the RhoB promoter to increase the expression level of RhoB, is a drug that induces apoptosis.

1 is a diagram showing the structure of the RhoB promoter located upstream of the luciferase gene in the pGL2-basic vector.

Figure 2 is a diagram showing a luciferase reporter system (pGL-basic-RhiB promoter vector) for the discovery of RhoB expression increasing substance of the present invention.

3 is a graph measuring the degree of RhoB promoter activation for each drug by the intensity of luciferase fluorescence. 1 to 6 on the Y axis represent the fluorescence intensity of the negative control group (CTL) cells as 1, and the fluorescence intensity is expressed in multiples. As a positive control, SAHA, FTI, and GGTI were treated with cells at 5 or 10 uM concentration to measure the intensity of fluorescence. NCI drugs were also treated with cells at 5 or 10 uM concentration to measure the intensity of fluorescence. X-axis 1 to 9 are the following materials. 1, NSC156272 (C21H19NO6.HCl); 2, NSC126188 (C22H45N2O.I); 3, NSC608781 (C28H36O3); 4, NSC205357 (C5H8N4Se); 5, NSC19703; 6, NSC 67586 (C7H16N2OS2); 7, NSC 233064 (C25H37Cl2N2O3P); 8, NSC287407 (C38H36O6); 9, NSC297364 (C19H17ClN2O).

Figure 4 is a schematic diagram showing a cyan fluorescent reporter system (pTagCFP-N-RhoB promoter vector) for the discovery of RhoB expression increasing substance of the present invention.

5 is a diagram showing the cyan fluorescence of the degree of RhoB promoter activation. DMSO without drug treatment, SAHA, and FTI-277 were used as a positive control drug, Sorafenib was used as a negative control drug, and NSC126188 (C22H45N2O.I) was used as a drug for measuring RhoB RhoB promoter activation. .

FIG. 6 is a photograph of dead cells and apoptosis-induced cells treated with Hela cell line with NSC 126188 and stained with annexin V and PI.

7 is a diagram showing the expression level of the case 3, its substrate, PARP, and RhoB through Western blot after treatment of Hela cells with NSC126188. Cyclin B protein expression was confirmed as a positive control.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> METHOD OF SCREENING APOPTOSIS INDUCING ANTICANCER REAGENTS USING          RHOB PROMOTER REPORTER SYSTEM <130> PA070667 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 595 <212> DNA <213> Homo sapiens <220> <221> promoter (222) (1) .. (595) <223> RhoB PROMOTER GENE <400> 1 gcagcggcag cagcagcgcg gactccccgg tcgctgcctc tcccagcccg gcggcctggg 60 ccgtcaatca agctggccct gccccgccct cgggctgcag ggggcggcca atcagagcta 120 agctccgcag cgatgagctc agccggctgg tttcccattg gacggctata ttaagaaagt 180 ggccggactc tttaaatagc gggcgctagg gccgcagccc tcatctgcca ccgcagtctg 240 gttggagctg ttgtcttgta tgctcagcga ggcccggaga gacccgggag agagctaggc 300 cgagtccacc gcccgagtct gctgcccgag cccgcgttac gcacaaagcc gccgatcccc 360 ggcctggggt gagcagagcg accaccgccc gggagcagcg cggcgagacg cacggtgcgc 420 cctatgcccc cgcgccccca ccgcccccgc cgcggcagcc gaagcgcagc gagagaacgc 480 gccaccgcgg ggcccgggtg cagctagcga ccctctcgcc acctgcgcgc agcccgaggt 540 gagcagtgag cggcgagcgg gagggcagcg aggcgttcgc gggccccctc ctgct 595 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> RhoB-Luciferase-FW <400> 2 attggtaccg gcagcggcag cagcagcgcg g 31 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> RhoB-Luciferase-RV <400> 3 atataagctt agcaggaggg ggcccgcgaa cgc 33 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RhoB-CFP-FW <400> 4 taatattaat cgcagcggca gcagcagcgc 30 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> RohB-Luciferase-RV <400> 5 atataagctt agcaggaggg ggcccgcgaa cgc 33  

Claims (6)

Iii) a nucleotide encoding a RhoB promoter, and Ii) A screening system of RhoB gene expression increasing substances comprising nucleotides encoding luciferase or cyan fluorescent protein (CFP) and comprising host cells transformed with plasmids that do not encode other promoters or enhancers. The screening system of claim 1, wherein the plasmid is shown in FIG. 2. The screening system of claim 1, wherein the plasmid is shown in FIG. 4. The screening system of claim 1, wherein the host cell is a cancer cell. The screening system of claim 4, wherein the cancer cells are HeLa cells. a) treating the screening system of any one of claims 1 to 5 with candidate candidates for controlling expression of the RhoB promoter, and b) measuring the fluorescence of said system.

Images