Detection method for DNA repair-related genes which respond to low-level radiation

Abstract

Disclosed is a detection method for DNA repair-related genes which respond to low-level radiation, which includes the steps of (1) breeding AKR/J mice, as a thymus cancer model, and normal ICR mice in a low-dose radiation environment; (2) collecting thymuses from the AKR/J thymus cancer mouse model and the normal ICR mice which have been bred at the step (1); (3) analyzing genes in the thymuses collected at the step (2); (4) detecting, among the genes analyzed at the step (3), a DNA repair-associated gene expressed commonly or differentially in the AKR/J thymus cancer mouse model and the normal ICR mice; and (5) amplifying the gene detected at the step (4) and measuring the expression level thereof.

Claims

1. A method of detecting a DNA repair-associated gene which responds to low-dose radiation, comprising the steps of: (1) breeding a thymus cancer mouse model and a normal mouse in a low-dose radiation environment, wherein the thymus cancer mouse model and the normal mouse are housed in a low-dose radiation environment of 0.7 mGy/h until a total cumulative dose reaches 1.7 Gy (gray); (2) collecting thymuses from the thymus cancer mouse model and the normal mouse which have been bred at the step (1); (3) analyzing genes in the thymuses collected at the step (2); (4) detecting, among the genes analyzed at the step (3), a DNA repair-associated gene expressed commonly or differentially in the thymus cancer mouse model and the normal mouse, wherein the DNA repair-associated gene is Ptgs2 (prostaglandin-endoperoxide synthase 2) gene; and (5) amplifying the gene detected at the step (4) and measuring an expression level thereof.

2. The method of claim 1, wherein, at the step (5), the expression level is measured by a Venn diagram, quantitative polymerase chain reaction, detection assay for a specific protein and a SAS statistical program.

3. A method of detecting a DNA repair-associated gene which responds to low-dose radiation, comprising the steps of: (1) breeding a thymus cancer mouse model and a normal mouse in a low-dose radiation environment, wherein the thymus cancer mouse model and the normal mouse are housed in a low-dose radiation environment of 0.7 mGy/h until a total cumulative dose reaches 1.7 Gy (gray); (2) collecting thymuses from the thymus cancer mouse model and the normal mouse which have been bred at the step (1); (3) analyzing genes in the thymuses collected at the step (2); (4) detecting, among the genes analyzed at the step (3), a DNA repair-associated gene expressed commonly or differentially in the thymus cancer mouse model and the normal mouse, wherein the DNA repair-associated gene is Rnd3 (Rho family GTPase 3) gene; and (5) amplifying the gene detected at the step (4) and measuring an expression level thereof.

4. The method of claim 3, wherein, at the step (5), the expression level is measured by a Venn diagram, quantitative polymerase chain reaction, detection assay for a specific protein and a SAS statistical program.

5. A method of detecting a DNA repair-associated gene which responds to low-dose radiation, comprising the steps of: (1) breeding a thymus cancer mouse model and a normal mouse in a low-dose radiation environment, wherein the thymus cancer mouse model and the normal mouse are housed in a low-dose radiation environment of 0.7 mGy/h until a total cumulative dose reaches 1.7 Gy (gray); (2) collecting thymuses from the thymus cancer mouse model and the normal mouse which have been bred at the step (1); (3) analyzing genes in the thymuses collected at the step (2); (4) detecting, among the genes analyzed at the step (3), a DNA repair-associated gene expressed commonly or differentially in the thymus cancer mouse model and the normal mouse, wherein the DNA repair-associated gene is Plxnc1 (plexin C1) gene; and (5) amplifying the gene detected at the step (4) and measuring an expression level thereof.

6. The method of claim 5, wherein, at the step (5), the expression level is measured by a Venn diagram, quantitative polymerase chain reaction, detection assay for a specific protein and a SAS statistical program.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a graph showing the relative expression levels of genes, among genes associated with DNA damage and repair, being species-specifically responsive to low-dose radiation, the expression being analyzed by polymerase chain reaction;

(2) FIG. 2 shows the protein expression levels in thymuses from AKR/J mice and ICR mice, both of which were exposed to low-dose radiation ((1) DNA repair-associated genes in response to low-dose radiation (Cyp11a1, Rnd3 and Plxncl), (2) loading control, and (3) quantitative analysis for proteins of interest, in which the density of each protein band was measured and quantified relative to the density of -tubulin); and

(3) FIG. 3 is a schematic diagram showing gene expression responses to low-dose radiation and subsequent cellular changes in the thymus from a mouse exposed to low-dose radiation.

BEST MODE

(4) Hereinafter, the present invention will be described in detail.

(5) Generally, it is known that high-dose-rate radiation causes DNA damage and eventually induces cancer. In contrast, low-dose-rate radiation is known to stimulate repair of damaged DNA, apoptosis, immune responses and antioxidant capacity so that cancer incidence is suppressed. However, many cases do not satisfy the low dose rate (6 mGy/h) radiation recommended by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR 2000). Also, the interpretation of the data obtained from cell lines is generally difficult to directly apply to humans. Moreover, even if experimental animals were used, there are no conventional reports that confirm the effects of low-dose radiation on DNA repair with reference to protein levels. In the present invention, AKR/J mice and normal mice (ICR) were reared under low-dose-rate (0.7 mGy/h) irradiation. When mice started to die (Day 100), thymuses were collected and analyzed for gene expression. Also, from normal mice (ICR) reared under the same conditions, thymuses were collected and analyzed for gene expression. Finally, identified were DNA repair-associated genes expressed commonly or differentially between the two species of mice, and the genes were amplified for protein detection, thus affording genomic and proteomic profiling.

Mode for Invention

(6) Hereinafter, the present invention will be explained in more detail with reference to the following examples. The following examples are provided only to illustrate the present invention, but it is to be understood that the present invention is not deemed to be limited thereto.

EXAMPLE 1

(7) Seven-week-old female AKR/J mice and ICR mice were purchased from Shizuoka laboratory Center (Japan) and maintained under specific pathogen-free conditions for an adaptation period of one week. Five mice in each group were housed in a low-dose-rate (0.7 mGy/h) radiation facility for a period of 100 days until the total cumulative dose reached 1.7 Gy. Thereafter, thymuses were collected, rapidly frozen in liquid nitrogen, and analyzed for gene expression.

(8) Gene expression patterns in the thymuses were compared between AKR/J mice and ICR mice, which were exposed to low-dose radiation. As a result, identified were DNA repair-associated genes responsive to low-dose-rate (0.7 mGy/h) radiation, and the genes were investigated for their functions.

(9) The genes were analyzed using a Venn diagram, quantitative polymerase chain reaction, detection assay for specific proteins and the SAS statistical program (ANOVA, t-test).

(10) For DNA repair-associated genes identified as radiation-responsive in thymuses from mice exposed to low-dose-rate (0.7 mGy/h) radiation, expression levels were analyzed using primers having nucleotide sequences listed in Table 1, below.

(11) TABLE-US-00001 TABLE1 Gene Forward Reverse accessionNo. Genes (5.fwdarw.3) (5.fwdarw.3) NM_019779 Cypllal CCTGGAAGAAAGACCG TGCTTGATGCGTCTGTG AATC(SEQIDNO:1) TAA(SEQIDNO:2) NM_011198 Ptgs2 AGAACCTGCAGTTTGC GCTCCTGCTTGAGTAT TGTG(SEQIDNO:3) GTCG(SEQIDNO:4) NM_028810 Pnd3 TATGACAACGTCCGTC CCTGGATTTCACCTTTC CACT(SEQIDNO:5) CAC(SEQIDNO:6) NM_018797 Plxncl TCCTCATCCCATGAAG CGCTGCTAAGCACTCT AACA(SEQIDNO:7) GAAC(SEQIDNO:8) NM_021282 Cyp2el TCTCTTCAACAAACGC CCAGGGAGTACTCAGC TTCG(SEQIDNO:9) AGGT(SEQIDNO:10)

TEST EXAMPLE 1

Measurement of Expression Levels of DNA Damage/Repair-Associated Genes

(12) After AKR/J mice and ICR mice were exposed to low-dose-rate (0.7 mGy/h) radiation, thymuses were collected at the early stage of carcinogenesis (Day 100). Focusing on genes associated with DNA damage and repair, genes species-specifically responsive to low-dose radiation were selected and analyzed for relative expression levels via polymerase chain reaction.

(13) As shown in FIG. 1, Cyp11a1, Ptgs2, Rnd3 and Plxncl genes in AKR/J mice irradiated with low-dose radiation and cyp2el gene in ICR mice were found to be species-specifically responsive to low-dose radiation.

TEST EXAMPLE 2

Evaluation for Protein Expression in Thymuses from Low-Dose Radiation-Irradiated AKR/J and ICR Mice

(14) Protein expression levels were estimated in the mouse thymuses prepared in Example 1, which were irradiated with low-dose radiation, along with a control group in which mice were exposed to high-dose radiation (0.8 Gy/min, the total dose: 4.5 Gy).

(15) As shown in FIG. 2, in the AKR/J mice irradiated with low-dose radiation, Cyp11a1, Rnd3 and Plxncl genes were identified as species-specifically responsive to low-dose radiation.

(16) Hereinbefore, the present invention has been described in detail with reference to specific examples thereof. Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention. Thus, the actual scope of the present invention will be defined by the appended claims and equivalents thereof.

(17) <Sequence Listing Free Text>

(18) The sequence listing was submitted as an electronic file.