KR100541529B1 - A marker for diagnosing radio-resistance or radiation-induced cancer, a kit or microarray for diagnosing radio-resistance or radiation-induced cancer containing the same, a screening method using the same - Google Patents

Abstract

The present invention relates to a radiation-resistant or radiation-oncology diagnostic marker comprising a nucleotide of NCBI Gene Bank Accession No. X58876 or S93521, a radiation-resistant or radiation-oncology diagnostic kit or microarray comprising the same. And a method for searching for a drug for treating or inhibiting radiation-resistant or radiation-carcinogenicity using the same.

Radiation-resistant, radiation-carcinogenic

Description

A marker for diagnosing radiation-resistant or radiation-carcinogenesis, a radiation-resistant or radiation-carcinogenic diagnosis kit or microarray including the same, and a method for searching using the same diagnosing radio-resistance or radiation-induced cancer containing the same, a screening method using the same}

1 shows the screening process of radiation-resistant clones,

2a and 2b show the results of measuring the survival rate of the radiation-resistant clone,

Figure 3 shows the tumor induction test results of radiation-resistant clones in nude mice,

Figure 4 shows the histological examination of the induced tumor,

5 shows the results of RT-PCR analysis,

6 shows Western blotting results for CDC25B and MDM2,

Figure 7 shows the results of radiation-resistant induction of CDC25B or MDM2 gene overexpressing cells,

Fig. 8 shows the tumor producing ability test results of CDC25B or MDM2 gene overexpressing cells.

The present invention relates to a radiation-resistant or radiation-carcinogenic diagnostic marker, a radiation-resistant or radiation-carcinogenic diagnostic kit or microarray comprising the same, and a method for searching for a drug for treating or inhibiting radiation-resistant or radiation-carcinogenic cancer using the same. will be.

Cancer treatment by radiation has been known as an essential treatment for the treatment of various types of cancer. However, such a radiation treatment method has problems such as a decrease in treatment efficiency due to acquiring resistance of cancer cells to radiation and generation of secondary cancer by radiation after radiation treatment. In particular, the problem of secondary cancer generation is estimated to be induced by gene mutations such as expression of oncogenic genes or inhibition of oncogenic gene expression by radiation after radiation treatment.

For example, it has been reported that radiation can cause cancers such as skin cancer and leukemia (Upton AC (1986) Historical perspective on radiation carcinogenesis: in Radiation Carcinotenesis, Upton AC, Alber RE, Burns FJ and Shore RE (eds) (pp1-10, Elsvier, New York), and radiation carcinogenesis studies using a large number of experimental animals such as mice and rats have shown evidence that radiation can cause cancer. The results have been attached and confirmed that radiation is a potent cancer-causing substance (Fry RJM and Storer JB (1987) External radiation carcinogenesis: in Advances in Radiation Biology, Lett JT (ed.) Vol 13, pp 31-90, Academic Press , New York). Among them, data on Hiroshima / Nagasa A-bomb victims suggest that radiation causes cancer in most tissues and at most ages.

However, due to the specificity of the radiation itself (physical factor), it is assumed that there will be a difference in cancer generation from other chemical carcinogens or viral carcinogens, but until now cancers induced by radiation and other causes The specific difference with is not confirmed.

In studies related to carcinogenesis by radiation, radiation is known to induce chromosomal alterations and cell inactivation, including gene mutations (Breimer LH (1988) Ionizing radiation-induced mutagenesis.Br J Cancer 57: 6-18) In addition, Krolewski B and Little JB (1995) Alterations of mdm2 gene in X-ray transformed mouse 10T1 / 2 cells clones.Int J Oncol, 6: 1123-1127; Vahakangas KH, Samet JM, Metcalf RA, Welsh JA, Bennett WP, Lane DP and Harris CC (1992) Mutation of p53 and ras genes in radon-associated lung cancer from uranium miners.Lancet 339: 576-580; Dutrillaux B (1997) Ionizing radiation induced malignancies in man.Radioprotection, 32: C431-C440), the exact mechanism for this phenomenon is not known.

Therefore, if the radiation-resistant gene is diagnosed and confirmed in advance, it will be possible to select the treatment method and predict the treatment effect in the radiation treatment. Thus, genes that exhibit resistance to radiation and induce oncogenesis are useful as markers for radiation-resistant diagnosis and for measuring radiation-carcinogenesis (eg, secondary cancer development by radiation).

The present invention has been made based on the above prior art, and the present inventors have completed the present invention by identifying genes involved in radiation-resistant and tumorigenicity through cDNA microarray analysis.

Accordingly, an object of the present invention is to provide a marker for diagnosing radiation-resistant or radiation-carcinogenicity.

It is also an object of the present invention to provide a radiation-resistant or radiation-oncology diagnostic kit comprising the marker.

It is also an object of the present invention to provide a radiation-resistant or radiation-carcinogenic diagnostic microarray comprising the marker.

It is also an object of the present invention to provide a method for searching for a drug for treating or inhibiting radiation-resistant or radiation-carcinogenicity using the radiation-resistant gene.

In order to achieve the above object of the present invention, the present invention provides a radiation-resistant or radiation-oncology diagnostic marker comprising nucleotides of NCBI Gene Bank Accession No. X58876 or S93521.

The present invention also provides a radiation-resistant or radiation-carcinogenic diagnostic kit comprising the marker and a radiation-resistant or radiation-carcinogenic diagnostic microarray comprising the marker.

In addition, the present invention comprises the steps of irradiating a mammal, except humans; Contacting the test material with cells extracted from the irradiated mammal; The present invention provides a method for searching for a drug for treating or inhibiting radiation-resistant or radiation-carcinogen, comprising measuring a change in expression of the radiation-resistant gene from the cell.

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

As used herein, the term "radio-resistance or radiation-resistance" refers to obtaining resistance to radiation by over-expression or less expression by irradiation, and "radiation-cancer ( The term "radiation-induced cancer" refers to overexpression or less expression by radiation to obtain resistance to radiation and to induce tumorigenesis, and for example, to generate secondary cancer by radiation.

The nucleotides useful as a radiation-resistant or radiation-carcinogenic diagnostic marker according to the present invention establish a cell line inducing resistance by radiation through cDNA microarray analysis to confirm tumor cellization, thereby obtaining resistance of cells to radiation and A group of genes involved in tumorization was identified.

That is, mouse fibroblasts (NIH3T3) were irradiated with gamma rays to select clones of surviving cells, and clones showing radiation resistance were selected through clonalogenic analysis. It was confirmed that tumors were generated by transplanting them into nude mice, and 20 genes showing expression differences with normal cells were identified by cDNA microarray analysis. The 20 genes are as follows: NCBIA gene bank grant no. AF132483 (cyclin-dependent kinase 6), JO4806 (secreted phosphoprotein 1), M87276 (thrombospondin 1), X58876 (mdm2), X52046 (procollagen, type III) , alpha 1), S38100 (vascular endothelial growth factor), AF077003 (CD2-associated protein), S93521 (cdc25 homolog B (S. cerevisiae)), L33726 (fascin homolog 1), X14194 (nidogen 1), U09659 (heat shock 10 kDa protein 1 (chaperonin 10)), M85078 (colony stimulating factor 2 receptor, alpha, low-affinity), X13358 (nuclear receptor subfamily 3, group C, member 1), M13177 (transforming growth factor, beta 1), Z14249 (mitogen activated protein kinase 3), U84411 (protein tyrosine phosphatase 4a1), Y00703 (guanine nucleotide binding protein, alpha stimulating), D30687 (glutathion S-transferase, pi 2), D78645 (heat shock 70kD protein 5 (glucose regulated protien, 78 kD)), and X62622 (tissue inhibitor of metalloproteinase 2).

RT-PCR of these cells confirmed that 10 genes showed the same results as the cDNA microarray test results, among which NCVIA Genebank grant No. X58876 (Dilla T, Romero J, Sanstisteban P, Velasco). JA (2002) The mdm2 proto-oncogene sensitizes human medullary thyroid carcinoma cells to ionizing radiation.Oncogene, 21: 2376-2386) and S93521 (Galaktionov K and Beach D. (1991) Specific activation of cdc25 tyrosine phosphatase by B-type cyclins : Evidence for multiple roles of mitotic cyclins.Cell, 67: 1181-1194 The ten genes containing the nucleotides of NCBI gene bank accession numbers X58876 and S93521 are as follows: NC gene gene bank accession numbers AF132483 (cyclin-dependent kinase 6), JO4806 (secreted phosphoprotein 1), X58876 (mdm2) , X52046 (procollagen, type III, alpha 1), AF077003 (CD2-associated protein), S93521 (cdc25 homolog B (S. cerevisiae)), L33726 (fascin homolog 1), X13358 (nuclear receptor subfamily 3, group C, member 1), Z14249 (mitogen activated protein kinase 3), and X14194 (nidogen 1).

Accordingly, the present invention includes a marker for diagnosing radiation-resistant or radiation-oncology comprising the nucleotides of NCBI Gene Bank Accession No. X58876 or S93521. In addition, the radiation-resistant or radiation-oncology diagnostic markers may further include nucleotides selected from the group consisting of NCVIA GenBank grant numbers AF132483, JO4806, X52046, AF077003, L33726, X13358, Z14249, and X14194. .

In addition, the present invention includes a radiation-resistant or radiation-carcinogenicity diagnostic kit comprising the marker. The present invention also includes a radiation-resistant or radiation-carcinogenic diagnostic microarray comprising the marker. Diagnostic kits and microarrays according to the invention can be prepared according to conventional methods used in the art using such radiation-resistant or radiation-carcinogenic diagnostic markers.

In addition, the present invention comprises the steps of irradiating a mammal, except humans; Contacting the test material with cells extracted from the irradiated mammal; And a method for searching for a drug for treating or inhibiting radiation-resistant or radiation-carcinogenicity, comprising measuring a change in the expression of a radiation-resistant gene comprising the nucleotides of NCBAIN Gen. No. X58876 or S93521 from the cell. do. In the above searching method, the radiation-resistant gene preferably further comprises nucleotides selected from the group consisting of NCBIN Gene Bank Accession Nos. AF132483, JO4806, X52046, AF077003, L33726, X13358, Z14249, and X14194.

In the search method according to the present invention, mammals that can be used include rats, mice, guinea pigs, rabbits, hamsters and the like. In addition, the expression change of the radiation-resistant gene can be measured using a kit or a microarray comprising a radiation-resistant or radiation-carcinogenic diagnostic marker according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples do not limit the present invention.

Example 1 Screening for Clones Showing Radiation Tolerance

5X10 ⁶ mouse fibroblasts (NIH3T3) were placed in a 60mm dish, incubated for 3 days in a 37 ° C CO ₂ incubator, and 4 Gy of gamma-ray ( ¹³⁷ Cs) (Atomic Energy of Canada, Ltd., Canada) was 3.81 Gy / min dose rate. 48 times for 6 months once every 3 days and incubated (see Figure 1). After confirming that about 200 or more colonies were formed, the mixture was fixed using a mixture of methanol and acetic acid (methanol: acetic acid = 3: 1) as a fixed solution, and stained with trypan blue to count the number of colonies to form colonies. As a result, clones (7 clones) of surviving cells were selected (FIG. 2A).

These clones were subjected to clonogenic analysis. That is, 500 cells were plated in 6 cm dishes (plating) and then irradiated with increasing radiation dose, and the culture was continued until colonies were formed. Once the appropriate colonies were formed, they were fixed with fixative (methanol: acetic acid = 3: 1) and then stained with trypan blue to count the number of colonies and the two clones with the highest resistance to radiation (NIH3T3-). R # 1 and R- # 4) were selected (FIG. 2B).

Example 2. Animal Testing

Mouse Tumor development occurred 6 weeks after subcutaneous transplantation of normal fibroblasts (NIH3T3) and clones obtained in Example 1 (NIH3T3-R # 1, NIH3T3-R # 4) 1x10 ⁶ per head subcutaneously in the back of the nude mouse (Balb C nude) It observed, and the result is as shown in FIG. As can be seen in Figure 3, when the normal fibroblasts (NIH3T3) transplanted tumors were not produced, it can be seen that the mice transplanted with NIH3T3-R # 1 and R- # 4.

The resulting tumor was removed to remove tumor tissue and then the tissue was fixed in 10% buffered formalin and embedded in paraffin blocks. Tissue sections were made to a size of 0.2 um, adhered to glass slides, and then stained with hematoxylin / eosin, followed by histological examination. The results are shown in FIG. 4. As can be seen in Figure 4, compared with the case of implantation of normal fibroblasts (NIH3T3) (A), tumors appear in common tumors in tumor tissues (B: clear cells, C: apoptotic body, D : Multinucleated cells, E: necrosis) can be observed.

Example 3. cDNA Microarray Analysis

CDNA microscopy of normal fibroblasts (NIH3T3) and resistant cells obtained in Example 1 (NIH3T3-R # 1 and R- # 4) to identify differences in gene expression of cells that induce radiation-resistant and tumorigenicity Array analysis was performed.

Using Triazol ^™ (Life Technology, Inc; Gaithersburg, MD, USA) Total RNA from normal fibroblasts (NIH3T3) and the resistant clones obtained in Example 1 (NIH3T3-R # 1, -R # 4) was isolated. Total RNA was prepared for ³² P-labeled cDNA probes by reverse transcriptation. Mouse cDNA microarray filters (Atlas Mouse Arrays membrane ™, 1176 genes, Clontech) were used for array hybridization.

cDNA microarray analysis revealed 20 genes showing differences in expression (increased expression in NIH3T3-R # 1 and R # 4) in both of the two resistant clones (NIH3T3-R # 1 and R- # 4). (19-expression increase, 1-expression decrease). Each gene is shown in Table 1.

Gene Bank License Number Gene name Overexpression AF132483 cyclin-dependent kinase 6 5.6 JO4806 secreted phosphoprotein 1 4.4 M87276 thrombospondin 1 3.2 X58876 mdm2 4.8 X52046 procollagen, type III, alpha 1 2.6 S38100 vascular endothelial growth factor 2.5 AF077003 CD2-associated protein 3.7 S93521 cdc25 homolog B (S. cerevisiae) 4.6 L33726 fascin homolog 1 3.0 X14194 nidogen 1 -2.5 U09659 heat shock 10 kDa protein 1 (chaperonin 10) 2.9 M85078 colony stimulating factor 2 receptor, alpha, low-affinity 2.6 X13358 nuclear receptor subfamily 3, group C, member 1 2.4 M13177 transforming growth factor, beta 1 3.3 Z14249 mitogen activated protein kinase 3 3.5 U84411 protein tyrosine phosphatase 4a1 2.9 Y00703 guanine nucleotide binding protein, alpha stimulating 2.7 D30687 glutathion S-transferase, pi 2 2.3 D78645 heat shock 70kD protein 5 (glucose regulated protien, 78kD) 2.7 X62622 tissue inhibitor of metalloproteinase 2 3.1

Example 4. RT-PCR Analysis

After total RNA was isolated in the same manner as in Example 3, reverse transcription was performed using 1 μg of total RNA. After preparing RNA with 1 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 the cDNA was synthesized at 37 ° C. for 1 hour, followed by 95 ° C. Revers Transcriptase was inactivated for 5 minutes at. The reaction mixture (RT mixture) was composed of 2.5 μl 10X buffer, 2.5 μl (uM) dNTP mixture, 1.25 μl oligo dT primer, 1.25 μl (U) reverse transcriptase, 0.5 μl RNasin.

PCR was performed using the synthesized cDNA. Each primer was designed to suit each mouse gene, and all were designed to have annealing temperature of 58 ° C. 2 μl of each cDNA was used, and the reaction mixture was added and denatured at 95 ° C. for 5 minutes, followed by 1 minute at 94 ° C. (denature), 1 minute at 58 ° C. (annealing), and 30 minutes at 72 ° C. (polymerization). After performing a cycle of PCR, the polymerization was performed again at 72 ° C. for 5 minutes. The reaction mixture (PCR mixture) contains 2 μl 10 × buffer, 2.5 μl (uM) dNTP mixture, 1 μl Forward Primer, 1 μl Reverse Primer, 0.1 μl (U) Taq DNA Polymerase (TaKaRa) , 11.4 μl distilled water.

As a result of the RT-PCR analysis, ten genes showed the same results as the cDNA microarray analysis of Example 3 (see FIG. 5). These ten genes are shown in Table 2 below.

Gene Bank License Number Gene name function AF132483 cyclin-dependent kinase 6 Cell cycle regulation JO4806 secreted phosphoprotein 1 Tumorigenesis, Tissueremodeling X58876 mdm2 Carcinogen X52046 procollagen, type III, alpha 1 Intercellular interaction AF077003 CD2-associated protein Actin cytoskleton generation S93521 cdc25 homolog B (S. cerevisiae) Cell cycle regulation L33726 fascin homolog 1 Create Actin bundle X13358 nuclear receptor subfamily 3, group C, member 1 Hormone metabolism regulation Z14249 mitogen activated protein kinase 3 Cell signaling X14194 nidogen 1 Intercellular interaction

From the RT-PCR results, Western blotting was performed on MDM-2 and CDC25B with the clearest difference in expression change. In other words, the protein was extracted from the cells with Lysis Buffer (Lysis Buffer), the protein was separated from the SDS PAGE and transferred to nitrocellulose. MDM-2. An antibody against CDC25B protein (Santa Cruz) was attached to the membrane, a horseradish peroxidase-linked secondary antibody was attached, and protein expression was confirmed using an ECL reagent (Enhanced Chemoluminescence, Amersham). , And the result is shown in FIG.

Example 5 Selection of Cells with Overexpression Vector Inserted

An expression vector (Dilla T, Romero J, Sanstisteban P, Velasco JA (2002) The mdm2 proto-oncogene sensitizes into which the pcDNA3 vector (Stratagene Co., Ltd.) inserted the genes of NCBAIN Genbank License No. X58876 (MDM2) or S93521 (CDC25B). human medullary thyroid carcinoma cells to ionizing radiation.Oncogene, 21: 2376-2386; and Galaktionov K and Beach D. (1991) Specific activation of cdc25 tyrosine phosphatase by B-type cyclins: evidence for multiple roles of mitotic cyclins.Cell, 67 : 1181-1194) were overexpressed in mouse fibroblasts (NIH3T3) using lipofectin (GIBCO, Gaithersburg, MD). Cells into which the expression vector was inserted were selected using G418 (GIBCO, Gaithersburg, MD), and three clones each having a high expression rate of MDM2 or CDC25B gene (MDM2 # 1, 2, and 3; CDC25B # 1, 3). , And 8) were selected.

Example 6 Measurement of Resistance to Radiation

Clonality survival assay (clonogenic survival assay) was performed in the same manner as in Example 1 to the radiation resistance of the clones selected in Example 5, the results are shown in FIG. As can be seen in Figure 7, it can be seen that the selected clones exhibit a degree of resistance similar to NIH3T3-R # 1 and -R # 4.

In addition, when 1x10 ⁶ cells of each of the clones selected in Example 5 were subcutaneously implanted and observed for 6 weeks, it can be seen that tumors were formed as NIH3T3-R # 1 and -R # 4. (Figure 8).

Radiation-resistant or radiation-carcinogenic diagnostic markers, kits and microarrays comprising the same according to the present invention can be usefully used to identify genes that exhibit resistance to radiation after radiation treatment and exposure and induce tumorigenesis. The search method according to the invention is useful for searching for drugs that treat or inhibit radiation-resistant or radiation-carcinogenicity.

Claims (6)

NCBI Gene Bank Accession No. X58876 or radiation-resistant or radiation-carcinogenic diagnostic markers comprising the nucleotides of S93521. The marker for diagnosing radiation-resistant or radiation-cancer according to claim 1, wherein NCVIA further comprises nucleotides selected from the group consisting of GenBank grant Nos. AF132483, JO4806, X52046, AF077003, L33726, X13358, Z14249, and X14194. Radiation-resistant or radiation-carcinogenicity diagnostic kit comprising the marker according to claim 1. A radiation-resistant or radiation-carcinogenic diagnostic microarray comprising the marker according to claim 1. Irradiating mammals other than humans; Contacting the test material with cells extracted from the irradiated mammal; And A method of searching for a drug for treating or inhibiting radiation-resistant or radiation-carcinogenicity, comprising measuring a change in the expression of a radiation-resistant gene comprising nucleotides of NCBAIN Gene Bank No. X58876 or S93521 from the cell. 6. The method of claim 5, wherein the radiation-resistant gene further comprises nucleotides selected from the group consisting of NCVIA GenBank grant numbers AF132483, JO4806, X52046, AF077003, L33726, X13358, Z14249, and X14194. Search method.

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