KR101808863B1 - Screening Methods of apoptotic inducer agents using ROR - Google Patents

Abstract

The present invention relates to a method for screening a cell death-inducing substance using RORα, which discloses a molecular mechanism involved in cell death, and provides a method for screening a cell death inducing substance using the molecular mechanism. (A) contacting a cell containing the RORa gene or protein with a test substance to be analyzed; And (b) confirming the increase of the RORα protein in the cell. The present invention also provides a screening method for inducing apoptosis.

Description

Screening methods of apoptotic inducer agents using ROR < RTI ID = 0.0 >

The present invention relates to a method for screening a cell death-inducing substance using ROR ?.

DNA damage response is an important defense mechanism against genotoxic stress and acts as a barrier to cancer progression by inducing cell cycle arrest, apoptosis, DNA repair and / or aging. The crucial factor in the DNA damage response is the p53 tumor suppressor protein, and cells with defective function of p53 develop into malignant tumors without regulating cell proliferation. Under normal conditions, the expression of p53 remains low through constitutive degradation by E3 ubiquitin ligase MDM2. On the other hand, under DNA damage, p53 is rapidly stabilized without MDM2-mediated degradation. Stabilization dependent on DNA damage and transcriptional activation of p53 also induces downstream target genes such as cell cycle arrest, apoptosis, and DNA repair associated with p21, Bax and Puma.

 p53 activity is regulated by protein stabilization, post-transcriptional modification, protein-protein interactions, and subcellular localization. Stabilization of p53 is important for the transcriptional activity of p53, and post-transcriptional modification of p53 is involved in regulation of p53 stabilization and function. The transcriptional activity of p53 regulates transcriptional coactivators or corepressors. Many proteins have been reported to bind to p53 and regulate transcriptional activity. SRY-associated HMG-box transcription factor family SOX4 increases p53 acetylation by inhibiting MDM2-mediated degradation, stabilizing p53 protein, and promoting p53 / p300 / CBP complex formation leading to transcriptional activation. UBE4B, on the other hand, induces p53 inactivation in brain cancer and promotes MDM2-mediated degradation of p53.

RORα is one of a family of orphan nuclear receptors. The RORα gene is often located in vulnerable areas and where frequent loss occurs, and RORα appears to play a role in cancer suppression. The present inventors have recently reported that RORa inhibits the Wnt /? -Catenin signaling pathway associated with cancer progression in colorectal cancer, thereby regulating cell proliferation and cancer progression.

Korean Patent Application No. 10-2010-0082161 discloses an anticancer agent screening method using RORα, comprising: culturing cells; Contacting the cell with a potential substance; Confirming that the phosphorylation of the intracellular RORa is increased relative to that of the control (the test sample not contacting the potential substance to the cell); And selecting the latent substance that increases phosphorylation of ROR alpha as an anticancer agent.

However, it is not known how RORα is associated with apoptosis induction. We have shown that DNA damage induces RORα, identifies functional p53 response factors in the RORα promoter, stabilizes p53 by preventing DNA damage-inducing RORα from p53 ubiquitination, and activates p53 transcription by the HAUSP-dependent method I found out. In addition, through analysis of the genome-wide microarray, we confirmed that RORα-mediated p53 stabilization activates a subset of p53 target genes specifically associated with apoptosis, and has been applied to the treatment of various diseases associated with apoptosis through RORα And completed the present invention.

The present invention discloses that the role of RORα, which has not been well known so far, is that RORα positively regulates p53, thereby increasing cell death and suppressing cancer, thereby screening the cell death-inducing substance using RORα The purpose is to provide.

The present invention relates to a method for screening a cell death-inducing substance using RORα, which discloses a molecular mechanism involved in cell death, and provides a method for screening a cell death inducing substance using the molecular mechanism. A first aspect of the present invention is a method for assaying a test compound comprising the steps of: (a) contacting a test substance to be analyzed with cells containing the RORa gene or protein; (b) confirming the binding of the RORα protein in the cell. The present invention also provides a screening method for inducing apoptosis. And more particularly to a method for screening for apoptosis inducing substance, which further comprises measuring the activation of a transcript of p53 in the cell. In another aspect of the present invention, there is provided a screening method for inducing apoptosis, which comprises further measuring the inhibition of ubiquitination of p53 in the cell. In another aspect of the present invention, there is provided a screening method for inducing apoptosis comprising the step of additionally measuring inhibition of ubiquitination of p53 in the cell. In another embodiment of the present invention, there is provided a screening method for inducing apoptosis, which comprises further measuring a decrease in ubiquitination of p53 in the cell. The assay method includes, but is not limited to, (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation or coimmunoprecipitation assays, immunodiffusion assays, complement fixation assays, (Complement Fixation Assay), FACS, and a protein chip may be used. The cells may be animal cells or animals, and may be Drosophila cells or Drosophila cells.

A second aspect of the present invention relates to a method for screening a test sample, comprising: (a) contacting a cell containing an RORa gene or protein with a test substance to be analyzed; And (b) confirming intracellular increase of the ROR alpha protein in the cell. In another aspect of the present invention, there is provided a screening method for a cancer therapeutic agent, which comprises further measuring activation of a transcript of p53 in the cell. In another aspect of the present invention, there is provided a screening method of cancer therapeutic agent, which comprises further measuring the inhibition of ubiquitination of p53 in the cell. In another aspect of the present invention, there is provided a screening method of cancer therapeutic agent, which comprises further measuring the inhibition of ubiquitination of p53 in the cell. In another aspect of the present invention, there is provided a method for screening a cancer treatment agent, which comprises further measuring a decrease in ubiquitination of p53 in the cell. The measurement method includes a tissue immuno staining, a radioimmunoassay (RIA ), Enzyme immunoassay (ELISA), Western blotting, immunoprecipitation or coimmunoprecipitation assays, immunodiffusion assays, complement fixation assays, FACS, And a protein chip. The present invention also relates to a method for screening a cancer therapeutic agent. The cell may be an animal cell or an animal, and may be a Drosophila cell or a Drosophila.

A third aspect of the present invention relates to a method for producing a test compound, comprising: (a) contacting a test substance to be analyzed with a cell comprising an RORα gene promoter and a reporter operably linked thereto; And

(b) confirming the intracellular increase of the RORα protein in the cell.

A fourth aspect of the present invention is a method for producing a test sample comprising the steps of: (a) contacting a cell containing a RORα gene promoter and a reporter operably linked thereto with a test substance to be analyzed; And

(b) identifying an intracellular increase of the ROR alpha protein in the cell.

The substance screened by the screening method of the present invention is a substance having apoptosis inducing activity and can be used as a therapeutic agent for various diseases and diseases that can be treated by inducing apoptosis. For example, diseases that can be treated by inducing apoptosis include cancer and fibrosis, which include, but are not limited to, liver fibrosis, pulmonary fibrosis, and renal fibrosis.

The present invention relates to a screening method of apoptosis-inducing substance using RORα, and a screening method of the present invention is a novel approach to easily and efficiently screen substances which are effective in inducing apoptosis and preventing or treating cancer to be. According to the present invention, it is possible to effectively screen lead compounds of new drugs inducing apoptosis, thereby improving the hit ratio in preclinical and clinical tests, and consequently, The cost and the time required to develop the system can be greatly reduced.

Fig. 1 shows the discovery of ROR? As a target gene of the p53 gene induced by DNA damage.
Figure 2 shows that RORa regulates the stabilization of p53 by inhibiting ubiquitination of p53.
Figure 3 shows that RORa increases deubiquitination of p53 in a HAUSP-dependent manner.
FIG. 4 shows that RORα regulates a subset of p53-responsive genes involved in apoptosis as a result of the genome analysis.
Figure 5 shows that RORa is introduced into a subset of the p53 target promoter with HAUSP.
Figure 6 shows that RORa increases apoptosis through p53 in response to DNA damage.
Figure 7 shows that RORa regulates IR- and p53-induced apoptosis in Drosophila.
Figure 8 shows a real-time RT-PCR analysis in RORa and p21 mRNA time- and dose-dependent manner and shows a comparison with p53 isoforms.
Figure 9 shows that RORa increases HAUSP-mediated p53 de-ubiquitination.
FIG. 10 shows real-time RT-PCR analysis of RORa-dependent p53 reactive gene.
Figure 11 shows that RORa increases cell death through p53 in response to DNA damage.
Figure 12 shows IR- and p53-induced cell death in Drosophila.

< Example  1> DNA  Damage mediated ROR alpha is p53 Effect on

To investigate the molecular mechanism of whether RORα is involved in tumor suppression, the present inventors tried to measure upstream signals through the induction of RORα. Since p53 is one of the most important factors in the signal pathway under the DNA damage reaction, the present inventors examined whether induction of RORα activates p53. As a result, induction of RORα activated the expression of p53, and loss of p53 failed to induce RORα in response to DNA damage, indicating that the induction of RORα was dependent on p53.

< Example  2> In the promoter p53  Containing a response factor p53  As a target gene ROR Identification of α

To investigate how RORα is up-regulated in response to DNA damage in dependence on p53, the inventors performed experiments on the assumption that RORα is a direct p53 target gene containing a functional p53 response factor (p53REs) in its promoter . As a result, DNA damage-mediated RORα induction was found to be p53-specific rather than p63 or p73 induced by RORα expression.

< Example  3> p53 Ubiquitination  Disturbed ROR of p53  Stabilization control

The inventors investigated the induction effect of ROR alpha in DNA damage. Simultaneous-immunoprecipitation analysis revealed that RORa and p53 interact at endogenous expression levels. In addition, we examined whether overexpression of RORα accumulates p53 protein, and the level of p53 protein dramatically increased according to overexpression of RORα. In order to determine whether RORα regulates p53 stabilization, the half-life of p53 protein was measured by cyclohexamide treatment, and the half-life of p53 protein was increased by overexpression of RORα.

Because p53 is degraded through the ubiquitin proteasome pathway, we have tested whether RORα affects p53 ubiquitination, resulting in a dramatic increase in p53 ubiquitination by RORα knockdown, which suggests that RORα interferes with p53 ubiquitination Lt; / RTI &gt;

< Example  4> genome Wide  Through gene expression analysis ROR alpha-dependent p53  Cell death of reactive genes ( apoptosis )

To identify p53 target genes that are affected by RORa under DNA damage, we performed WT or RORa-deficient Staggerer MEFs gene expression analysis with and without Dox treatment. When Dox was treated, the expression levels of 3,861 genes were significantly compared in WT or ROR alpha-deficient Staggerer MEFs. Analysis showed that the regulation of apoptosis was due to potential RORα-dependent p53 target genes.

< Example  5> DNA  In the damage response p53 through ROR Increased cell death of α

RORa regulates DNA damage-mediated apoptosis. As a result, it was found that RORα promotes p53-mediated cell death in response to DNA damage, and RORα-induced cell death is dependent on p53.

Transcriptional activation of p53 is related to cell cycle arrest, apoptosis, and DNA repair. RORα stabilizes p53 and activates it to induce apoptosis, thereby increasing or stabilizing RORα expression level. Can be an effective anticancer agent and can efficiently screen anticancer agents using this mechanism and can be applied to the development of a therapeutic agent for various diseases related to apoptosis mechanism. Table 1 lists the genes that are independent of ROR alpha dependent and ROR alpha in 9 clusters. Table 2 lists previously known p53 reactive genes for insertion into clusters.

Claims (18)

(a) contacting a test substance with an animal cell comprising an RORa gene or protein;
(b) measuring the expression level of the RORα protein in the cell; And
(c) screening the test substance as a cell death inducing substance when the expression of the RORα protein is increased in a cell contacted with the test substance as compared with a control group not in contact with the test substance, Way.
The method according to claim 1,
The method comprising the further step of measuring an increase in the expression of p53 in the cell, wherein when the expression of the p53 is increased in cells contacted with the test substance as compared with the control group not in contact with the test substance, Lt; RTI ID = 0.0 &gt; induction. &Lt; / RTI &gt;
The method according to claim 1,
The method according to any one of Claims 1 to 5, further comprising the step of measuring the inhibition of ubiquitination of p53 in the cell, wherein, when the ubiquitination of the p53 is inhibited in cells contacted with the test substance as compared to a control group not in contact with the test substance, Is selected as a cell death inducing substance.
The method according to claim 1,
Wherein the animal cell is a Drosophila cell or a Drosophila adult.
The method according to any one of claims 1 to 4, wherein the cell death inducing substance is used as an anticancer agent. delete delete delete delete delete delete delete delete delete delete delete delete delete

Images