# Patent

## Title
DNP63A gene and screening methods of anticancer agent by using it

## Abstract

This invention relates to a gene encoding ΔNp63α and screening methods of anticancer-drugs thereof, more specifically a gene encoding ΔNp63α and a protein which is transported from nucleus to cytoplasm by contacting with potential anti-cancer-drugs in an epithelial cell carcinoma, a recombinant vector comprising said gene and reporter genes, and carcinoma cells comprising said vector. Also, This invention relates to high throughput screening methods of anticancer-drug comprising identifying the transportation of ΔNp63α protein from nucleus to cytoplasm by contacting with potential anticancer-drug in a carcinoma cell.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. §371 from international application PCT/KR2007/001576, filed Mar. 30, 2007.
TECHNICAL FIELD
The function of ΔNp63α which is specifically expressed in the epidermal stem cells and cancer cells and repressed during differentiation could be very important in the point of that about 80% of human cancer is derived from epithelial cell and cancer could be derived by genetic aberration from stem cell. Also as ΔNp63α has a function to inhibit p53 in cell proliferation and death and works as a carcinogenesis control gene when it is over-expressed, the quantitative balance between p53 and ΔNp63α in stem cell is regarded to control cellular proliferation and differentiation. Therefore, ΔNp63α's stability in protein level and its location in cell are thought to exert important function in the carcinogenesis.
p63 gene, located on chromosome 3q, encodes a nuclear protein with high sequence similarity with p53. p53 protein is a tumor-suppressor, having a transcription activation domain which is important in the regulation of cell cycle and cell death. Protein p63 exists in two different forms, each produced from distict transcripts by two selective promoters. The first transcript is translated to full-length proteins including N-terminal transcription activation domain like p53. It can cause cell death. The second transcript is translated to truncated proteins lacking N-terminal transcription activation domain (ΔNp63). ΔNp63 suppresses transcriptional activity of p53 and/or full-length p63 in a dominant-negative manner.
BACKGROUND ART
US Patent Publication No. US20020094547A1 (Invention Title: Differential diagnosis of cancer and other conditions based on expression of p63) relates to a gene product, referred to herein as p63, and to nucleic acids, including the gene, mRNAs, and cDNAs, encoding it, in particular, to methods of using the gene and gene product to diagnose and/or treat certain diseases and disorders such as cancer. The invention also relates to kits which may be used in such diagnostic and treatment methods. The invention also provides methods of distinguishing an epithelial squamous cell carcinoma from a non-epithelial cell carcinoma by detecting p63 nucleic acid or protein expression in cells derived from a carcinoma. In particular, p63 expression indicates that the carcinoma is an epithelial squamous cell carcinoma and the absence of p63 expression indicates that the carcinoma is a non-epithelial cell carcinoma or a carcinoma without squamous differentiation potential.
PCT Publication No. WO04048520A2 (Invention Title: A Transcriptional Target of p63 and p53, and method of use therefor) relates to identification of a novel shared transcriptional target, termed REDD1, that implicates ROS in the p53-dependent DNA damage response and in p63-mediated regulation of epithelial differentiation is disclosed.
EA Patent No. EA0003326B1 (Invention Title: Methods of cancer treating) relates to a method of treating a human subject for cancer comprising the steps of: (a) administering to said subject an organic non-peptide compound that is capable of binding to one or more domains of a human protein of the p53 family under physiological conditions, and stabilizing a functional conformation therein, and (b) permitting said stabilized protein to interact with one or more macromolecules that participate in a wild-type activity of said protein.
U.S. Pat. No. 6,479,285 (Invention Title: p53 as a regulator of cell differentiation) provides methods of the blocking of p53 function in embryonic tissues, and the use of these tissues as screening tools for substances that are capable of overcoming the p53-related block in differentiation. However, there is no example to screen for anti-cancer drugs using biochemical characters of ΔNp63α and exchange of intracellular location.
The present inventors found that ΔNp63α protein was transported from nucleus to cytoplasm during degradation, thereby completing the present invention.
DISCLOSURE
Technical Problem
It is an object of the present invention to develop an effective anticancer-drug screening system in a short time using characters of ΔNp63α, the specific growth regulator of epithelial cell carcinoma.
Technical Solution
The present invention relates to a gene encoding a ΔNp63α transported from the nucleus to the cytoplasm due to contact with potential anticancer-drugs in an epithelial cell carcinoma.
The present invention relates to a ΔNp63α protein transported from the nucleus to the cytoplasm due to contact with potential anticancer-drugs in an epithelial cell carcinoma.
The present invention relates to a ΔNp63α gene transported from the nucleus to the cytoplasm due to injection with potential anticancer-drugs in an epithelial cell carcinoma, and a recombinant vector comprising reporter genes to identify said gene.
The present invention relates to an epithelial cell carcinoma transformed with said recombinant vector.
The present invention relates to screening methods of anticancer-drugs comprising identifying the transportation of a ΔNp63α protein from the nucleus to the cytoplasm due to injection with potential anticancer-drugs in an epithelial cell carcinoma.
The present invention relates to screening methods of anticancer-drugs comprising that said carcinoma is transformed with a ΔNp63α gene and a recombinant vector comprising reporter genes to identify said gene.
The present invention relates to screening methods of anticancer-drugs comprising the steps of:
Cloning a ΔNp63α gene and reporter genes to identify said gene into a vector;
Transforming an epithelial cell carcinoma with said cloning vector and expressing for a certain time;
Contacting said carcinoma with potential materials; and
Identifying the transportation of a ΔNp63α gene location from the nucleus to the cytoplasm.



DESCRIPTION OF DRAWINGS
FIG. 1 shows that the amount of ΔNp63α is decreased in the protein level after TNF-α treatment. After TNF-α treatment for 16 hours, cyclohexamide which inhibit protein synthesis was added and the samples were obtained at the indicated time-points and observed for changes of protein stability. As a result, the protein degradation began after 4 hours of treatment with cyclohexamide. And treating with the proteasome inhibitor, MG132 (20 nM) together, the protein wasn't degraded and stabilized even after 4 hours.
FIG. 2 shows the transportation of the ΔNp63α protein from the nucleus to the cytoplasm after TNF-α treatment.
FIG. 3 shows a cleavage map of a recombinant expression vector comprising a ΔNp63α and a reporter gene according to the present invention.



BEST MODE
According to a first aspect, the present invention provides the gene of SEQ ID NO: 1 encoding the ΔNp63α protein which is transported from nucleus to cytoplasm by contacting with potential anticancer-drug in a carcinoma cell derived from epithelial cell. Preferably, cDNA of SEQ ID NO: 2, which resulted in reverse transcription of ΔNp63α mRNA, is used.
As used herein, the term “potential anticancer-drug” refers to effective materials that can kill cancer cells.
According to a second aspect, the present invention provides the ΔNp63α protein of SEQ ID NO: 2, which is transported from nucleus to cytoplasm by contacting with potential anticancer-drug in a carcinoma cell derived from epithelial cell.
According to a third aspect, the present invention provides a expression vector comprising a ΔNp63α gene which is transported from nucleus to cytoplasm by injection with potential anticancer-drug in a carcinoma cell derived from epithelial cell and a reporter gene to identify the expression in of said gene.
As used herein, the term “vector” means a nucleic acid molecule that can carry another nucleic acid bound thereto. As used herein, the term “expression vector” is intended to include a plasmid, cosmid or phage, which can synthesize a protein encoded by a recombinant gene carried by said vector. A preferred vector is a vector that can self-replicate and express a nucleic acid bound thereto.
According to a fourth aspect, the present invention provides a carcinoma cell derived from epithelial cell, which is transformed with said recombinant vector.
As used herein, the term “transformation” means that foreign DNA or RNA is absorbed into cells to change the genotype of the cells.
A suitable carcinoma cell derived from epithelial cell includes HeLa cells, HaCAT, ZR-75-1 or ME180, but is not limited thereto.
According to a fifth aspect, the present invention provides a method of screening for anticancer-drug comprising a step of identifying the transportation of a ΔNp63α protein from nucleus to cytoplasm by injection with potential anticancer-drug in a carcinoma cell derived from epithelial cell.
Methods for identifying the transportation to cytoplasm can be to use reporter genes. But reporter genes are not limited to the specific gene. Any genes that expressed easily detectable products can be used. Suitable reporter genes are well known to those of skill in the art. Examples of reporter genes include other enzyme detection systems such as chloramphenicol acetyl transferase (CAT), luciferase and β-galactosidase, bacterial luciferase, alkaline phosphatase and green fluorescent protein (GFP), but are not limited thereto. Detection of reporter genes is well known to those of skill in the art. In the case of the reporter gene detected by measuring the enzyme activity, the present invention includes steps of supplying with the enzyme and the suitable substrate generally, and detecting reaction products (for example, light produced by luciferase). Also, the present invention can include steps of detecting the presence or absence of gene products easily. Alternatively, the present invention can include quantification of expression level of reporter gene products. Methods for quantification refer to absolute quantification or quantification relative to the expression level of the housekeeping gene. These assays can be performed manually or using high-throughput systems automatically.
High throughput assays for the presence, absence, or quantification of gene expression are well known to those of skill in the art. For example, U.S. Pat. No. 5,559,410 discloses high throughput screening methods for protein and U.S. Pat. No. 5,585,635 discloses high throughput screening methods for nucleic acids binding. Also, U.S. Pat. Nos. 5,576,220 and 5,541,061 describe methods for ligand/antibody binding.
According to a sixth aspect, the present invention provides the method of screening for anticancer-drug comprising the steps of:
Cloning a ΔNp63α gene and a reporter gene to identify the expression of said gene into a vector;
Transforming with said cloned vector into a carcinoma cell derived from epithelial cell and expressing the ΔNp63α gene and the reporter gene;
Contacting said carcinoma cell with potential anticancer-drug; and
Identifying the transportation of the ΔNp63α gene location from nucleus to cytoplasm.
According to a seventh aspect, the present invention provides a kit for performing said screening methods.
Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.
MODE FOR INVENTION
Reference Example
Previously, ME180, ΔNp63α overexpressing carcinoma, was treated with TNF-α (tumor necrosis factor-alpha) which induces cell death. Also, the changes of the amount of ΔNp63α protein were observed. As a result, the amount of ΔNp63α decreased rapidly after treatment of TNF-α. It is identified that such degradation of ΔNp63α was proceeded by specific mechanism, that is, by proteasome-mediated mechanism (FIG. 1). Protein degradation pathways could be usually classified in large into two groups, the first one is a degradation pathway by a protease after its polyubiquitination and movement to proteases, and the other one is a degradation pathway by a caspases.
Example 1
Preparation of Recombinant Vectors
A vector pEGFP-C1 (clontech) was used. The pEGFP-C1 vector is easy to detect with fluorescence in case it is expressed because it encodes EGFP (Enhanced green fluorescent protein) gene as a reporter gene. Fusion protein was constructed by subcloning of ΔDNp63α gene of SEQ ID No: 1, the target gene for expression, by using restriction sites of SalI and BamHI within the MCS (multiple cloning site) located at the 3′ end of the EGFP gene.
Example 2
Cell Preparation by Transformation with the Recombinant Vector
After preparation of competent E. coli DH10B cells using calcium chloride, transformation of E. coli was induced with the subcloned pEGFP-ΔNp63α by heat shock methods. At this time, the kanamycin was used as a selectable marker. The resulting colonies were incubated and then their plasmid DNAs were extracted with Miniprep Kit (Intron). The DNA sequence of the extracted plasmid DNAs were analyzed by the DNA sequence analysis.
Example 3
Methods of Screening for Anticancer Drugs
The recombinant vector constructed by the present inventor had fused ΔNp63α next to pEGFP-C1. The advantage of GFP is to observe easily in microscopy because GFP emit the green light at the specific absorbance (Excitation maximum=488 nm; emission maximum=507 nm). Therefore, the carcinoma cell line ME180 which was transformed with subcloned pEGFP-ΔNp63α by using Fugene 6 (Roche) was observed at 488 nm wavelength. GFP is inherently expressed all over the area of cell, however, it was identified that GFP expression was observed only in the nucleus after GFP fused with ΔNp63α. It was observed with microscope by mounting into a mounting solution having DAPI, after TNF-α (tumor necrosis factor-alpha) was treated and then cells were fixed timely.
As a signal of cell death induction, the intracellular location and expression patterns of ΔNp63α were observed. In detail, it was observed variation aspect and cell location of ΔNp63α with a time span, 0 hr, 1 hr, 2 hr and 4 hr with fluorescence microscope, after cloning ΔNp63α gene into the vector expressing Green Fluorescence Protein (GFP), expressing for a certain time by transformation into carcinoma cell, treating TNF-α which induce cell death, carcinoma cells being transformed and then the protein being expressed for a certain time. As a result, it was observed similar aspect of aggreasome, which looks like complex of preteasome, because ΔNp63α, which had been known to locate only in nucleus, moved from nucleus to cytoplasm about 2 hr after treatment of TNF-α (FIG. 2). Therefore, it could be expected that the degradation of ΔNp63α protein is conducted by proteasome in cytoplasm because its location change from nucleus to cytoplasm happen in a short time when cell death is induced.
Industrial Applicability
With a biochemical character of ΔNp63α which exchanges intracellular location, high-throughput screen for anticancer-drug could be achieved than the screening method of anticancer drug in the prior art. The present invention could be used to construct screening system for searching various anticancer drugs except TNF-α used herein, which induce carcinoma specific cell death.

## Claims

1. A method of screening candidate anticancer drugs comprising the steps of:
(a) contacting an epithelial carcinoma cell with a candidate anticancer drug; and
(b) identifying the transportation of ΔNp63α protein from nucleus to cytoplasm upon contact with the candidate anticancer drug.

2. The method according to claim 1, wherein the carcinoma cell is transformed with a recombinant vector expressing ΔNp63α and a a reporter gene to identify expression of ΔNp63α.

3. A method of screening candidate anticancer drugs comprising the steps of:
(a) transforming an epithelial carcinoma cell with a vector expressing ΔNp63α and a reporter gene;
(b) contacting said carcinoma cell with a candidate anticancer drug; and
(c) identifying the transportation of ΔNp63α protein from nucleus to cytoplasm in the carcinoma cell upon contact with the candidate anticancer drug.