KR20030095821A - A gene for measuring the degree of irradiation, a method for measuring the degree of irradiation using the same, and a DNA chip containing the same - Google Patents

Abstract

PURPOSE: A gene for measuring the degree of irradiation, a method for measuring the degree of irradiation using the same, and a DNA chip containing the same gene are provided, thereby improving the accuracy, sensitivity and time for measuring the degree of irradiation. CONSTITUTION: A gene for measuring the degree of irradiation is selected from the gene bank accession nos. af016266, 142176, m15796 and u53328. A method for measuring the degree of irradiation comprises measuring the expression increase of the gene selected from gene bank accession nos. af016266, 142176, m15796 and u53328 in a blood sample obtained from a person. The DNA chip contains one or more genes selected from gene bank accession nos. af016266, 142176, m15796 and u53328 as a biomarker.

Description

Gene for measuring radiation exposure, method for measuring radiation exposure using same, and DNA chip containing same {A gene for measuring the degree of irradiation, a method for measuring the degree of irradiation using the same, and a DNA chip containing the same}

The present invention relates to a gene for measuring radiation exposure, a method for measuring radiation exposure using the same, and a DNA chip containing the same as a biomarker, and more specifically, gene bank accession no. Af016266, 142176, m15796, and a gene for measuring radiation exposure selected from the group consisting of u53328, a method for measuring radiation exposure using the same, and a DNA chip containing the same as a biomarker.

Cancer treatment can be divided into surgery, radiotherapy, and chemotherapy. Currently, about 35% of cancer patients in Korea and about 50% in the United States are receiving radiation therapy. Since the trend is increasing every year, the importance of radiation therapy in the treatment of cancer is also increasing. However, as with chemotherapy, the biggest side effect of radiation therapy is that not only cancer cells but also normal cells are damaged by radiation. Therefore, radiotherapy should be treated to promote the death of cancer cells and minimize damage to normal cells. Therefore, it is very important to monitor the radiation exposure of normal cells during radiation therapy.

Monitoring the radiation exposure of normal cells is important not only in radiation therapy, but also for the diagnosis and treatment of radiation exposure in radiation-related workers who are at risk of radiation exposure.

The sensitivity to radiation according to cancer cells depends on the gene expression pattern, which is pointed out as one of the biggest problems in radiation therapy. Therefore, a method of monitoring the radiation response gene group of tumor cells and monitoring the radiation response gene group has been developed (Hanna E, Shrieve DC, Ratanatharathorn V, Xueqing X, Breau R, Suen J and Li). S: A novel alternative approach for prediction of radiation response of squamous cell carcinoma of head and neck.Cancer Res, 61: 2376-2380, 2000; Achary MP, Jaggernauth W, Gross E, Alfieri A, Klinger HP, Vikram B: Cell lines from the same cervical carcinoma but with different radiosensitivities exhibit different cDNA microarray patterns of gene expression.Cytogenet Cell Genet. 91 (1-4): 39-43, 2000; Galloway AM, Allalunis-Turner J: 4.cDNA expression array analysis of DNA repair genes in human glioma cells that lack or express DNA-PK.Radiat Res. 154 (6): 609-15, 2000). Among the damages caused by radiation is apoptosis, and these methods discovered a gene that induces radiation cell death using a DNA chip or the like.

Currently, efforts are being made to determine the difference in tumor cell responsiveness to radiation and to determine it (Amundson SA, Do KT, Shahab S, Bittner M, Meltzer P, Trent J, Fornace AJ Jr: Identification of potential mRNA biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation.Radiat Res. 154 (3): 342-6, 2000; Bump EA, Braubhut SJ, Lalayoor ST, Medeiros D, Lai LL, Cerce BA, Langley RE and Coleman CN: Novel concepts in modification of radiation sensitivity.Intl J Radiat Oncol Biol Phys, 29, 249-253, 1994; Hall EJ: Radiobiology for the radiologist. Lippincott, 3rd edition, p357-445, 1988), efforts to measure the degree of radiation exposure of normal cells. They are rarely done.

Genes that are known to be involved in radiation sensitivity are known as H-Ras oncogene c-Myc, Thymidine kinase, Basic fibroblast growth factor, Insulin-like growth factor, p21, Bcl-2, p53, Acid Sphinigomyelinase, and myeloid In cancer cells, p21WAF1 and GADD45 genes have been reported to respond between 2-50 cGy (FitxGerald TJ, Santucci MA, Das I, Kase K, Pierce JH and Greenberger JS: The v-abl, c -fms, or v-myc oncogene induces gamma radiation resistance of hematopoietic progenitor cell line at clinical low dose rate.Int J Radiat Oncol Biol Phys 21: 1203-1210, 1991; Riva C, Lavielli JP, Reyt E, Brambilla E, Lunardi J, Brambilla C: Differential c-myc, c-jun, c-far and p53 expression in squamous cell carcinoma of the head and neck: implication in drug and radioresistance.Eur J Cancer 31B: 384-391, 1995).

On the other hand, the method of measuring the radiation exposure is to isolate the lymphocytes of blood to check for chromosomal abnormalities, to measure micronucleus formation, to measure the mutation of glycoporin A in red blood cells, the degree of DNA cleavage, and the enamel of the tooth. Depends on the method of EST (Electron Spin Resonance), etc., but there are disadvantages in that it is different in reactivity, and it is almost impossible to measure low dose radiation and takes a long time (Brooks A: Biomarkers of exposure, sensitivity and Int J Radiat Biol, 75: 1481-1503, 1999; Amundson SA, Bittner M, Meltzer P, Trent J and Fornace AJ: Induction of gene expression as a monitor of exposure to ionizing radiation.Radiat Res, 156: 657- 661, 2001). In addition, in order to measure the exposure of biological radiation, measuring the degree of exposure by a single method is dangerous, and therefore, a method for improving accuracy is required.

Therefore, the present inventors have completed the present invention by developing a method for measuring the degree of radiation exposure and a DNA chip containing the same by selecting a group of genes that show a significant difference in sensitivity to radiation in normal cells.

Accordingly, it is an object of the present invention to provide a group of genes that exhibit marked differences in sensitivity to radiation.

It is also an object of the present invention to provide a method for measuring radiation exposure comprising the step of measuring the degree of expression increase of the gene group.

It is also an object of the present invention to provide a DNA chip comprising the gene group as a biomarker.

1 shows 44 gene groups with increased expression more than two-fold compared to controls not exposed to radiation,

Figure 2a and Figure 2b shows the results of the radioactivity of human blood lymphocytes tested by RT-PCR for 44 genes with increased expression in Example 1,

3 shows the expression level for four genes whose expression is increased by radiation,

4 shows the expression level of four genes whose expression is increased by radiation by expanding the number of subjects,

5a and 5b show the results of RT-PCR analysis of the expression level of the four genes over time and the radiation dose,

6a and 6b show the results of RT-PCR analysis of the expression level of the four genes in normal people who are not exposed to radiation.

To achieve the above object, the present invention provides a gene for radiation exposure measurement selected from the group consisting of gene bank accession numbers af016266, 142176, m15796, and u53328.

In addition, the present invention is a radiation exposure comprising the step of measuring the expression level of one or more genes selected from the group consisting of gene bank accession number (f016266, 142176, m15796, and u53328) of blood collected from normal people Provided is a measurement method, and preferably, when the degree of expression increase of the gene is detected to be at least about twice the mean expression value, it is determined that the radiation exposure measurement method characterized in that it is exposed to radiation.

The present invention also provides a DNA chip for measuring radiation exposure comprising a biomarker of at least one gene selected from the group consisting of gene bank accession nos af016266, 142176, m15796, and u53328.

Hereinafter, the present invention will be described in more detail.

Using 2400 gene chips, the present inventors separated and irradiated blood lymphocytes (PBLs) from healthy adults, and then total RNA was isolated and subjected to microarray analysis. Forty-four genes with more than two-fold increase in expression compared to unpopulated cells were selected (FIG. 1).

RT-PCR (Reverse Transcription-Polymerase Chain Reaction) analysis of 44 genes from five lymphocytes was performed to select four genes with more than double expression, such as microarray, in three or more of five humans. 2a and 2b. In addition, RT-PCR analysis of 30 or more normal subjects revealed that the four genes exhibited over 89% reactivity. In addition, these genes were also responsive by low dose radiation of 0.5 Gy, and the reactivity was continued until 24 hours of radiation exposure.

The finally selected genes are gene bank accession nos af016266, 142176, m15796, and u53328, which are known genes as TRAIL-receptor2, DRAL, Cyclin protein gene, and Cyclin G, respectively. It has a function as a dragon gene.

Therefore, radiation exposure can be measured by measuring the expression level of one or more genes selected from the group consisting of gene bank accession nos af016266, 142176, m15796, and u53328 in blood collected from normal individuals, Preferably, when the degree of expression increase of the gene is detected at least about twice the mean expression value, the radiation exposure can be easily measured by determining that it is exposed to radiation.

In addition, one or more genes selected from the group consisting of the gene bank accession numbers af016266, 142176, m15796, and u53328 may be used as biomarkers for preparing DNA chips for measuring radiation exposure. The DNA chip can be easily produced using a DNA chip manufacturing technique known in the art and using the gene according to the present invention as a biomarker.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited thereto.

(Example)

Example 1

Microarray Analysis

1. Selection of cell lines used

Peripheral Blood Lymphocytes (PBLs) were isolated from healthy adults.

2. Irradiation

PBLs were isolated and plated in 10 cm diameter Petri dishes and incubated at 37 ° C. for 2-4 hours. The gamma ray ¹³⁷ Cs (Atomic Energy of Canada, Ltd., Canada) was then investigated at a dose rate of 3.81 Gy / min. A total of 1 Gy was irradiated and RNA was isolated from PBL after 12 hours of irradiation.

3. Isolation of Total RNA

Isolation of total RNA was done using commercialized Triazol ^™ (Life Technology, Inc; Gaithersburg, MD, USA). MRNA was isolated from total RNA extracted from PBL isolated from 100 ml blood, and oligotex beads (Qiagen Co .; Santa Clarita, CA, USA) were used.

4. Microarray Analysis

Microarray analysis proceeded in three steps: labeling, hybridization, and washing.

Labeling was performed using 25 μl of reaction mixture. The reaction mixture consisted of 2 μl dNTP / primer mixed concentrate, 2 μl non-labeled control RNA, 20 μg (X μl) total RNA or mRNA, 13-X μl RNase-free water. The reaction mixture was treated for 10 minutes at 65 ° C. and 5 minutes at 25 ° C. before addition of 4 μl cyanine 3-dUTP or 2 μl cyanine 5-dUTP. Thereafter, the mixture was treated at 42 ° C. for 3 minutes, and then mixed with 2.5 μl 10 × reaction mixture buffer. 2 μl AMV RT / RNase inhibitor was added and incubated at 42 ° C. for 60 minutes, and then left at 4 ° C. for 10 minutes. 2.5 μl of 0.5M EDTA was added to stop the reaction, and 2.5 μl of 1N NaOH was added to initiate hydrolysis. Incubated at 65 ° C. for 30 minutes and 4 ° C. for 5 minutes, and neutralized with 6.2 μl of 1M Tris HCl (pH 7.5). Spin column (Spin column, Millipore Co .; Bedford, MA) was used to purely separate the resulting cDNA. cDNA was labeled with Cy-5 (red) and Cy-3 (green) to compare expression levels.

5. Microarray Scanning and Data Analysis

A NEN MICROMAX cDNA microarray was used and the NEN web page (www.nen.com) was referenced. Microarrays containing fluorescent images were scanned with a PIX 4000 microarray scanner (Axon Instruments) and image analysis was performed by a Gene PIX program. Genes that increased or decreased expression more than two-fold compared to unirradiated cells were considered as genes that respond to radiation.

6. Experimental Results

As a result of performing microarray analysis, FIG. 1 shows 44 gene groups in which expression is increased more than two times compared to the control group not exposed to radiation.

Representative functions of each gene increased expression of genes involved in various functions such as apoptosis, signaling, and mycochondrial function.

Example 2

RT-PCR Analysis

In Example 1, the following experiment was performed on 44 gene groups in which expression was increased more than two times compared to the control group not exposed to radiation.

1. RT-PCR Analysis

After 12 hours of 1 Gy irradiation, total RNA was isolated from PBL, and reverse transcription was performed using 1.25 μg of total RNA. After preparing RNA with 1.25 μg (X μl) total RNA and 17-X μl RNase-free water, the reaction was performed at 65 ° C. for 5 minutes, the reaction mixture was added, and cDNA was synthesized at 37 ° C. for 1 hour. Reverse Transcriptase was inactivated for 5 minutes. The reaction mixture consisted of 2.5 μl 10 × buffer, 2.5 μl dNTP mixture (5 mM each), 1.25 μl oligo dT primer (500 μg / ml), 1.25 μl reverse transcriptase (4 U / μl), 0.5 μl RNasin (40 U / Μl).

PCR was performed using the thus synthesized cDNA. Each primer was designed to suit each human gene, and all were designed to have an annealing temperature of 58 ° C. 2 μl of each cDNA was used, the reaction mixture was added and denatured at 95 ° C. for 5 minutes, followed by 95 ° C. 1 minute (denatured), 58 ° C. 1 minute (annealed), and 72 ° C. 1 minute (polymerization). After 30 cycles of PCR, polymerization was again performed at 72 ° C. for 6 minutes. The reaction mixture consisted of 2 μl 10X buffer, 2.5 μl dNTP mixture (2.5 mM each), 1 μl forward primer, 1 μl forward primer, 0.1 μl Taq DNA polymerase (TaKaRa 5 U / μl) , 11.4 μl distilled water.

2. Experimental Results

As a result of confirming the responsiveness to 1Gy radiation by RT-PCR using 5 normal blood of 44 genes, only 4 genes among 44 genes showed the same result as Example 1 in 3 or more bloods. The four genes were gene bank accession no af016266 (TRAIL-receptor 2), 142176 (DRAL gene), m15796 (Cell cycle protein), and u53328 (Cylcin G), and trail-receptor 2 (TRAIL- In all five patients for receptor 2), three out of five for DRAL gene, four out of five for Cell cycle protein, and five for Cyclin G gene Example 1 Showed the same result as (FIGS. 2A and 2B).

In addition, as a result of observing the expression level of the four genes, all four genes showed a two-fold increase in expression compared to control lymphocytes not irradiated (FIG. 3). Therefore, if the radiation exposure is diagnosed more than two times, it can be diagnosed as radiation exposure.

Example 3

Extended test

The RT-PCR test was carried out in the same manner as in Example 2 by expanding the number of subjects. Trail-receptor 2 was found in 29 (32%) of the total 32, and in 44 in the case of DRAL. In 39 (89%), 37 to 34 (92%) of cell cycle protein and Cyclin G increased expression by 1 Gy of radiation, resulting in microarray analysis. The same result was shown (FIG. 4).

As a result, the four genes can be used as a biomarker for diagnosing the exposure by showing 89% or more reactivity to 1Gy of radiation.

Example 4

Dose dependence of gene expression time and radiation dose

RT-PCR was performed at 12, 24 and 48 hours after radiation exposure to determine how long the expression of marker genes persisted after exposure and how long they could be measured until they were exposed to less radiation dose. Radiation doses were carried out with irradiation at 0.5, 1, 2 and 4 Gy, respectively.

The test results are shown in FIGS. 5A and 5B. As can be seen in Figures 5a and 5b, the radiation dose reactivity was well seen in the 12 hours of exposure, but after 24 hours the dose reactivity was shown for the cyclin protein gene and Cyclin G Trail-receptor 2 showed no difference between 0 and 5Gy and 4Gy, and DRAL responded only at relatively high doses (2 and 4Gy). After 48 hours, there was no difference in response between TRAIL-receptor 2 and DRAL, but the response was 0.5 Gy for cell cycle protein and Cyclin G. There was no difference.

Example 5

Expression difference measurement of marker genes in non-irradiated people

The expression level of the four marker genes was measured using the RT-PCR method of Example 2 in 12 people who were not exposed to radiation.

The results are shown in Figures 6a and 6b. Since the difference of the individual change was less than 2 times, it can be seen that four markers can be used as biomarkers upon radiation exposure. Since people who have been exposed to radiation cannot obtain normal blood without exposure, the level of expression before radiation exposure should not vary from person to person for use as a radiation exposure marker. Therefore, the above result with the difference of individual change of less than 2 times enables the four types of marker genes expressed more than 2 times compared to the control group can be used as an indicator for measuring the degree of radiation exposure.

According to the results of the above embodiments, the gene group consisting of four genes of TRAIL-receptor 2, DRAL, Cyclin protein, and Cyclin G, When exposed to radiation, the expression increased more than two times, and when not exposed to radiation, the difference in expression by individual was not more than doubled. It is considered that the method of measuring the radiation exposure degree using such a gene group is more preferable in terms of sensitivity, speed, and accuracy than other conventional methods.

As described above, according to the method of measuring radiation exposure using the gene for measuring radiation exposure of the present invention, it is possible to measure the radiation exposure of radiation more accurately and with low dose because the sensitivity is good and the measurement time is short. Can be. This helps to minimize side effects in the treatment of cancer and can help to maintain health by measuring radiation exposure of radiation workers more quickly and sensitively. In addition, using the gene chip of the present invention can easily predict and diagnose the degree of exposure to radiation in a radiation-related accident.

Claims (4)

Gene bank accession no. Gene for radiation exposure measurement selected from the group consisting of af016266, 142176, m15796, and u53328. 16. A method of measuring radiation exposure comprising measuring the level of expression increase of one or more genes selected from the group consisting of gene bank accession nos af016266, 142176, m15796, and u53328 in blood taken from a normal person. The method of measuring radiation exposure according to claim 2, wherein when the increase in expression level of the gene is detected to be at least about twice the mean expression value, it is determined that the radiation is exposed to radiation. Gene bank accession number (gene bank accession no) Af016266, 142176, m15796, and DNA chip for measuring radiation exposure comprising at least one gene selected from the group consisting of u53328 as a biomarker (biomarker).