KR100962185B1 - Marker genes based on daunorubicin treatment for screening of drug inducing cardio toxicity and screening method using thereof - Google Patents

Abstract

The present invention relates to a marker gene for cardiovascular toxicity-inducing drug search and a search method using the same, and more particularly, to a marker gene in which gene expression is increased or decreased by cardiovascular toxicity-inducing drug Daunorubicin, and a cardiovascular system using the same. The present invention relates to a method of searching for a toxic drug. The marker gene of the present invention can be usefully used for monitoring and determining drugs or chemicals having a risk of cardiovascular toxicity by using the response genes selected through the DNA microarray chip as marker genes. It can be used as a tool to identify mechanisms of action that cause side effects.

Daunorubicin, marker gene, microarray, cardiovascular toxicity

Description

Marker genes based on daunorubicin treatment for screening of drug inducing cardio toxicity and screening method using according to Daunorubicin treatment

The present invention relates to a marker gene for cardiovascular toxicity-inducing drug search and a search method using the same, and more particularly, to a marker gene whose expression is increased or decreased by daunorubicin, a cardiovascular toxicity-inducing drug, and daunorubicin using same. The present invention relates to a method for searching for a cardiovascular-induced drug.

Daunorubicin is used as a treatment for acute leukemia. Daunorubicin used in a narrow range is Streptomyces cis Percy Precious (Streptomyces in the early 1960 peucetius ) is commercialized and belongs to antitumor antibiotics.

Side effects of daunorubicin include myocardial disorders, ECG, tachycardia, heart failure, shock, anemia, granulocyte loss, thrombocytopenia, bleeding tendency, fever, rash, elevated liver function, kidney failure, ulcerative stomatitis, anorexia nervosa, Nausea, vomiting, diarrhea, hair loss, malaise, headache, dizziness, chills, shortness of breath, amenorrhea, atherosclerosis, and acquired resistance continue to develop, reducing drug influx into cancer cells and increasing drug outflow. It is resistant to drugs.

The chemical structural mechanism of daunorubicin is the first, which binds to DNA and is inserted into two double chains, and then destroys the DNA (a-helix) structure and prevents it from acting as a template for nucleic acid synthesis. Second, it has a DNA cutting effect due to the action of topoisomerase II or free radical formation (Line Wergelasdl., Et al ., BioMed Cental 1-11, 2007). Despite the side effects of daunorubicin in the human body, there is not enough risk assessment data in the human body, and the detection method for exposure is limited to the classical methods. It is an important task to discover and utilize molecular indicators that can be explored to perform appropriate measures and management for treatment and diagnosis using daunorubicin.

Several genome sequencing projects, including six mammals and 292 microbes, have been completed and reported to the National Center for Biotechnology Information (NCBI). Genome-wide expression studies are being conducted to study the function of genes based on the vast amount of data obtained. DNA microarray analysis is performed to analyze the expression of thousands of genes in a single experiment (Schena, M., et. al ., Proc . Natl . Acad . Sci . USA 93: 10614-10619, 1996).

Microarrays integrate a set of oligonucleotides of complementary DNA (cDNA) or 20-25 base pairs in length on glass. cDNA microarrays are produced by labs in schools or by companies such as Agilent and Genomic Solutions, by mechanically immobilizing cDNA collections on chips or by using ink jetting (Sellheyer, K and Belbin, TJ, J. Am . Acad . Dermatol . 51: 681-692, 2004). Oligonucleotide microarrays are made by Affymetrix using photolithography directly on the chip. (Sellheyer, K. et. Al., Am . Acad . Dermatol . 51: 681-692, 2004).

To analyze gene expression, RNA is obtained from samples such as tissues and hybridized with oligonucleotides in DNA microarrays. The obtained RNA is labeled with fluorescence or isotope and converted to cDNA. Oligo microarrays are mainly labeled with two different fluorescences (e.g., Cye3 and Cye5) to perform hybridization reactions on the same chip at the same time by labeling the control and experimental groups, respectively, and then optically scanning the images to obtain fluorescence intensity and analyzing the results. . Gene expression is determined according to the ratio of the two fluorescence intensities (Somasundaram, K., et al ., Genomics Proteomics I: 1-10, 2002).

In combination with recent research on toxic genomics (Toxicogenomics), an advanced technique using DNA macroarray technology, the expression pattern of genes expressed in specific tissues or cell lines by all chemicals as well as drug and new drug candidates in high throughput. Analysis and quantitative analysis are now possible. Accordingly, by analyzing the frequency of expression of specific genes in specific cells, it is possible to discover genes related to side effects of drugs, and through this, we will understand the molecular mechanisms according to the actions and side effects of drugs, Search and diagnosis of the material will be possible.

Therefore, the present inventors observed and analyzed gene expression profiles of daunorubicin in HUVEC cell lines, which are human umbilical vascular endothelial cells, using oligomicroarrays in which 41,000 human genes were accumulated. The present invention has been completed by establishing a marker gene capable of detecting daunorubicin and discovering a cardiovascular toxicity-inducing drug using the same.

Disclosure of Invention An object of the present invention is to provide a marker gene overexpressed or underexpressed by daunorubicin, a cardiovascular toxicity-inducing drug, and a cardiovascular toxicity-inducing drug search method using the marker gene.

In order to achieve the above object, the present invention provides a marker gene for cardiovascular toxicity-induced drug search characterized in that the expression changes in human umbilical vascular endothelial cells stimulated by cardiovascular-induced drug daunorubicin.

The present invention also provides an oligonucleotide comprising all or part of the marker gene gene sequence, or a DNA microarray chip for cardiovascular-induced drug search integrated with its complementary strand molecule.

In addition, the present invention provides a method for cardiovascular toxicity-induced drug search using the marker gene.

In addition, the present invention provides a kit for cardiovascular toxicity-inducing drug search comprising the DNA microarray chip.

Marker gene for cardiovascular toxicity-inducing drug search of the present invention and a cardiovascular toxicity-inducing drug search method using the same as a marker gene using a response gene selected through the DNA microarray chip drug or chemicals with a risk of new cardiovascular toxicity It is useful for monitoring or determining risk and can be used as a tool to identify mechanisms of action that cause cardiovascular toxicity and side effects caused by daunorubicin.

Hereinafter, the present invention will be described in detail.

The present invention provides a marker gene for cardiovascular toxicity-induced drug search, characterized in that the expression changes in human umbilical vascular endothelial cells stimulated by daunorubicin, a cardiovascular-induced drug.

The marker genes include apoptosis, cell cycle, DNA metabolism, regulation of protein kinase activity, regulation of transferase activity and alcohol metabolism. metabolism).

The present invention provides a marker gene, characterized in that selected from the group:

Genebank No. NM_078487 [cyclin-dependent kinase inhibitor 2b (p15, inhibits cdk4)], Genebank No. X06374 (platelet-derived growth factor alpha polypeptide), Genebank No. AK057315 [testis specific a2 homolog (mouse)], gene bank number AK127033 (dynamin 2), gene bank number (Genebank) AB209596 (g protein pathway suppressor 1), gene bank number (Genebank) AB209095 [cell division cycle 2-like 1 (pitslre) proteins)], Gene Registration Number (Genebank) AF033122 (sestrin 1), Gene Registration Number (Genebank) NM_053056 (cyclin d1), Gene Registration Number (Genebank) AY123223 (Sestrin 2), Gene Registration Number (Genebank) AK001361 [Protein phosphatase 1, regulatory (inhibitor) subunit 15A], Genebank NM_003550 [MAD1 mitotic arrest deficient-like 1 (yeast)], Genebank NM_139314 (Angiopoietin-like 4), Genebank (Genebank) NM_078467 [Cyclin-dependent kinase inhibitor 1A (p21, Cip 1)], Gene Registration Number (Genebank) NM_003311 (Pleckstrin homology-like domain, family A, member 2), Gene Registration Number (Genebank) AK125880 (Tumor protein p53 inducible nuclear protein 1), Gene Registration Number (Genebank) AB208907 ( Macrophage erythroblast attacher), Genebank AB096256 [Unc-5 homolog B (C. elegans)], Genebank AK092970 [protein (peptidylprolyl cis / trans isomerase) nima-interacting 1], Genebank CR623038 (inhibitor of dna binding 2, dominant negative helix-loop-helix protein), Genebank (Genebank) NM_033238 (promyelocytic leukemia), Genebank (Genebank) NM_003236 (transforming growth factor, alpha), Genebank (Genebank) BC060797 [par-6 partitioning defective 6 homolog gamma (c. Elegans)], Gene Registration Number (Genebank) NM_078467 [cyclin-dependent kinase inhibitor 1a (p21, cip1)], Gene Registration Number (Genebank) AY123223 (sestrin 2), Gene Registration Number (Genebank) NM_004642 (cdk2-associated protein 1), Gene Registration Genebank NM_033030 [bol, boule-like (drosophila)], Genebank NM_004359 (cell division cycle 34), Genebank CR612719 (growth arrest and dna-damage-inducible, alpha), Genebank (Genebank) BC064996 [rho guanine nucleotide excha nge factor (gef) 1], genebank NM_002838 (protein tyrosine phosphatase, receptor type, c), genebank NM_006455 (synaptonemal complex protein sc65), genebank AK074112 (cyclin l2) ), Genebank (Genebank) BM462208 (proliferating cell nuclear antigen), Genebank (Genebank) NM_02041 [poly (rc) binding protein 4], Genebank (Genebank) NM_006396 (sjogren's syndrome / scleroderma autoantigen 1), gene Genebank NM_003620 (protein phosphatase 1d magnesium-dependent, delta isoform), Genebank CR590366 [midkine (neurite growth-promoting factor 2)], Genebank NM_057158 (dual specificity phosphatase 4) (Genebank) AK001361 [protein phosphatase 1, regulatory (inhibitor) subunit 15a], gene registration number (Genebank) NM_002419 (mitogen-activated protein kinase kinase kinase 11), gene registration number (Genebank) NM_013258 (pyd and c ard domain containing), Genebank NM_013366 (anaphase promoting complex subunit 2), Genebank NM_012127 (cdkn1a interacting zinc finger protein 1), Genebank BC040303 (protein tyrosine phosphatase type iva, member 1), Genebank NM_002487 (necdin homolog (mouse)], Genebank (Genebank) NM_001759 (cyclin d2), Genebank (Genebank) NM_002632 (placental growth factor, vascular endothelial growth factor-related protein Genebank NM_005343 (v-ha-ras harvey rat sarcoma viral oncogene homolog), Genebank (Genebank) BC006795, M92424 [mdm2, transformed 3t3 cell double minute 2, p53 binding protein (mouse)], Genebank (Genebank) NM_003550 [mad1 mitotic arrest deficient-like 1 (yeast)], Genebank (Genebank) AK074652 (serta domain containing 1), Genebank (Genebank) BC094797 [unc-84 homolog b (c. elegans)], genebank (Genebank) CR612719 (growth arrest and dna-damage-inducible, alpha), genebank (Genebank) NM_181805 [protein kinase (camp-dependent, catalytic) inhibitor gamma], genebank (Genebank) ) M15169 (adrenergic, beta-2-, receptor, surface), Genebank AY247738 [tribbles homolog 3 (drosophila)], Genebank number AK074652 (serta domain containing 1), Gene registry number (Genebank) ) NM_139314 (angiopoietin-like 4), gene registration number (Genebank) BX641114 (annexin a4), gene registration number (Genebank) NM_002744 (protein kinase c, zeta), gene registration number (Genebank) R612719 (growth arrest and dna- damage-inducible, alpha), gene registration number (Genebank) BM557864 (heat shock 27kda protein 1), gene registration number (Genebank) NM_001024807 [amyloid beta (a4) precursor-like protein 1], gene registration number (Genebank) NM_003311 ( pleckstrin homology-like domain, family a, member 2), Genebank N M_020418 [poly (rc) binding protein 4], gene registration number (Genebank) AF332558 (bcl2 binding component 3), gene registration number (Genebank) NM_003811 [tumor necrosis factor (ligand) superfamily, member 9], gene registration number (Genebank) ) AK125880 (tumor protein p53 inducible nuclear protein 1), Genebank AK128462 (cisplatin resistance related protein crr9p), Genebank AK001361 [protein phosphatase 1, regulatory (inhibitor) subunit 15a], gene registration number (Genebank) AB208907 (macrophage erythroblast attacher), Genebank AB209361 [fas (tnf receptor superfamily, member 6)], Genebank NM_013258 (pyd and card domain containing), Genebank (Genebank) AB096256 [unc-5 homolog b (c. elegans), Genebank NM_004309 [rho gdp dissociation inhibitor (gdi) alpha], Genebank (Genebank) BC063507 (heat shock 70kda protein 1a), Genebank BG033621 (tumor protein, translationally- controlled 1), Genebank NM_003841 (tumor necrosis factor receptor superfamily, member 10c, decoy without an intracellular domain), Genebank NM_003806 [harakiri, bcl2 interacting protein (contains only bh3 domain)], gene Genebank CR611266 (fas-activated serine / threonine kinase), Gene Registration Number (Genebank) AK126489 (tubulin, beta), Gene Registration Number (Genebank) NM_003820 [tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry mediator) Genebank AK122614 (peroxisome proliferative activated receptor, delta), gene registration number AB020641 [PFTAIRE protein kinase 1], gene registration number AB033068 [Fizzy / cell division cycle 20 related 1 (Drosop) hila)], gene accession number AB057597 (Testis-specific kinase 2), gene accession number AB188493 (MAP / microtubule affinity-regulating kinase 2), gene accession number AB208919 [Fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer) syndrome)], gene accession number AB209154 (Receptor tyrosine kinase-like orphan receptor 2), gene accession number AB209179 [Polo-like kinase 1 (Drosophila)], gene accession number AB209337 [CDC7 cell division cycle 7 (S. cerevisiae)], gene registration number AB209615 (Protein phosphatase 5, catalytic subunit), gene registration number AB209681 [Rap guanine nucleotide exchange factor (GEF) 4], gene registration number AF041210 [Midline 1 (Opitz / BBB syndrome)], gene registration Number AF045459 (BMX non-receptor tyrosine kinase), gene registration number AF053305 [BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast)], gene registration number AF053306 [BUB1 budding uninhibited by benzimidazoles 1 homolog beta (yeast)], gene registration number AF332220 [Transducin (beta) -like 1Y-linked], gene accession no. AF375884 (Protein O-fucosyltransferase 1), gene accession no. AK000581 [Unc-51-like kinase 4 (C. elegans)], gene accession no. AK055221 (F- box protein 5), gene accession number AK056691 [Germ cell associated 2 (haspin)], gene accession number AK074377 (Chromosome 19 open reading frame 31), gene accession number AK091289 (Zinc finger protein 367), gene accession number AK092352 (Weakly similar to serine / threonine protein kinase Kp78), gene accession number AK123004 (Microtubule associated serine / threonine kinase-like), gene accession number AK131531 [Cyclin-dependent kinase (CDC2-like) 10), gene accession number AL833316 [Membrane-associated ring finger (C3HC4) 8], gene accession number AY257469 [Citron (rho-interacting, serine / threonine kinase 21)], gene accession number BC011498 (Histone deacetylase 6), gene accession number BC015416 (Ring finger protein 32), gene accession number BC032677 (Ubiquitin) -conjugating enzyme E2C), gene registration number BC035407 [Interleukin 3 receptor, alpha (low affinity)], gene registration number BC035514 [TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous and mucosal)], gene registration number BC038295 (Mitogen- activated protein kinase kinase 7), gene registration number BC043502 [never in mitosis gene a) -related kinase 2], gene registration number BC070168 (Tetraspanin 8), gene registration number BC071593 (V-kit Hardy-Zuckerman 4 feline sarcoma v iral oncogene homolog), gene accession number BC081541 [Poly (ADP-ribose) polymerase family, member 12], gene accession number BX537987 (Tripartite motif-containing 59), gene accession number CR749621 (SNF related kinase), gene accession number CR933728 ( Cell division cycle 2, G1 to S and G2 to M), gene registration number DQ097177 (HECT, UBA and WWE domain containing 1), gene registration number NM_000513 [Opsin 1 (cone pigments), medium-wave-sensitive (color blindness, deutan)], gene registration number NM_001005862 [V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro / glioblastoma derived oncogene homolog (avian)], gene accession number NM_001014796 (Discoidin domain receptor family, member 2), gene accession number NM_001274 CHK1 checkpoint homolog (S. pombe)], gene registration number NM_001490 [Glucosaminyl (N-acetyl) transferase 1, core 2 (beta-1,6-N-acetylglucosaminyltransferase)], gene registration number NM_001543 [N-deacetylase / N-sulfotransferase (heparan glucosaminyl) 1 ], Gene registration number NM_001618 [Poly (ADP-ribose) polymerase family, member 1], gene registration number NM_001789 (Cell division cycle 25A), gene registration number NM_001790 (Cell division cycle 25C), gene registration number NM_001798 (Cyclin-dependent kinase 2), gene registration number NM_002612 (Pyruvate dehydrogenase kinase, isozyme 4), gene registration number NM_002648 (Pim-1 oncogene), gene registration number NM_002758 (Mitogen-activated protein kinase kinase 6), gene registration number NM_002834 [Protein tyrosine phosphatase , non-receptor type 11 (Noonan syndrome 1)], gene registration number NM_002839 (Protein tyrosine phosphatase, receptor type, D), gene registration number NM_002843 (Protein tyrosine phosphatase, receptor type, J), gene registration number NM_002982 [Chemokine ( CC motif) ligand 2], gene registration number NM_003242 (Transforming growth factor, beta receptor II (70 / 80kDa)), gene registration number NM_003318 (TTK protein kinase), gene registration number NM_003335 (Ubiquitin-activating enzyme E1-like), gene Accession number NM_003384 (Vaccinia related kinase 1), gene accession number NM_003607 [CDC42 binding protein kinase alpha (DMPK-like)], gene accession number NM_004318 (Aspartate beta-hydroxylase), gene accession number NM_004667 (Hect domain and RLD 2), Gene registration number NM_004687 (Myotubularin related protein 4), gene registration number NM_004938 (Death-associated protein kinase 1), gene registration number NM_005082 (Tripartite motif-containing 25), gene registration number NM_005188 [Cas-Br-M (murine) ecotropic retroviral transforming sequence], gene accession number NM_005391 (Pyruvate dehydrogenase kinase, isozyme 3), gene accession number NM_005483 [Chromatin assembly factor 1, subunit A (p150)], gene accession number NM_005983 [S-phase kinase-assoc] iated protein 2 (p45)], gene registration number NM_006904 (protein kinase, DNA-activated, catalytic polypeptide), gene registration number NM_007039 (protein tyrosine phosphatase, non-receptor type 21), gene registration number NM_007295 (Breast cancer 1, early onset), gene accession number NM_007313 (V-abl Abelson murine leukemia viral oncogene homolog 1), gene accession number NM_007375 (TAR DNA binding protein), gene accession number NM_014176 [Ubiquitin-conjugating enzyme E2T (putative)], gene accession number NM_014238 (Kinase suppressor of ras 1), gene accession number NM_014264 [Polo-like kinase 4 (Drosophila)], gene accession number NM_014501 (Ubiquitin-conjugating enzyme E2S), gene accession number NM_014502 [PRP19 / PSO4 pre-mRNA processing factor 19 homolog (S. cerevisiae)], gene accession number NM_014671 (Ubiquitin protein ligase E3C), gene accession number NM_014791 (Maternal embryonic leucine zipper kinase), gene accession number NM_015148 (PAS domain containing serine / threonine kinase), gene accession number NM_015967 [Protein tyrosine phosphatase, non-receptor type 22 (lymphoid)], gene registration number NM_016513 [Intestinal cell (MAK-like) kinase], gene registration number NM_016653 (Hypothetical protein LOC339751), gene registration number NM_020205 (Zinc finger, A20 domain containing 1), gene Accession number NM_020438 (Dolichyl pyrophosphate phosphatase 1), gene accession number NM_020914 (Chromosome 17 open reading frame 27), gene accession number NM_021643 [Tribbles homolog 2 (Drosophila)], gene accession number NM_022455 (Nuclear receptor binding SET domain protein 1), Gene Registration No. NM_033389 [Slingshot homolog 2 (Drosophila), Gene Registration No. NM_080836 (Serine / threonine kinase 35), Gene Registration No. NM_153498 (Calcium / calmo dulin-dependent protein kinase ID), gene accession number NM_172207 (Calcium / calmodulin-dependent proteinkinase kinase 1, alpha), gene accession number NM_173216 (ST6nbeta-galactosamide alpha-2,6-sialyltranferase 1), gene accession number NM_198268 (Homeodomain interacting protein kinase 1), gene registration number U67156 (Mitogen-activated protein kinase kinase kinase 5), gene registration number (Genebank) AB033337 (M-phase phosphoprotein 1), gene registration number (Genebank) AB187578 [Shugoshin-like 1 (S . pombe), Genebank AB209271 (Ets2 repressor factor), Genebank (Genebank) AF345347 (Sperm associated antigen 5), Genebank AJ421269 (Regulator of chromosome condensation 2), Gene registry number ( Genebank) AK091170 [Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4)], gene accession number (Genebank) AK096393 (DIRAS family, GTP-binding RAS-like 3), gene accession number (Genebank) AK096541 (M-phase phosphoprotein 9), Genebank AL713771 (Nuclear transcription factor Y, gamma), Genebank AL832666 [Establishment of cohesion 1 homolog 2 (S. cerevisiae)], Genebank AL833052 (Hypothetical protein FLJ40137), Genebank AL833191 [SMC2 structural maintenance of chromosomes 2-like 1 (yeast)], Genebank AY366508 (Loss of heterozygosity, 11, chromosomal region 2, gene A), Gene registry number (Genebank) AY367065 [Asp (abnormal spindle)- like, microcephaly associated (Drosophila)], Genebank BC004352 (Kinesin family member 22), Genebank BC011000 (Cell division cycle associated 5), Genebank BC033086 [Transcription factor 19 ( SC1)], genebank BC038666 (Fanconi anemia, complementation group D2), genebank (Genebank) BC050369 (E2F transcription factor 1), genebank BC073878 (Kinesin family member C1), gene Genebank BX640816 (Nibrin), Genebank BG641109 [Spindle assembly 6 homolog (C. elegans), Genebank No. CR592757 [Barren homolog 1 (Drosophila)], Genebank No. D79987 [Extra spindle poles like 1 (S. cerevisiae)], Genebank No. NM_000059 (Breast cancer) 2, early onset, Genebank NM_000189 (Hexokinase 2), Genebank NM_001005414 (ZW10 interactor antisense), Genebank NM_001012271 (Effector cell peptidase receptor 1), Gene accession number (Genebank) Genebank) NM_001254 [CDC6 cell division cycle 6 homolog (S. cerevisiae)], gene accession number (Genebank) NM_001761 (Cyclin F), gene accession number (Genebank) NM_001813 (Centromere protein E, 312kDa), gene registration number (Genebank) NM_001827 (CDC28 protein kinase regulatory subunit 2), Genebank NM_002388 [MCM3 minichromosome maintenance deficient 3 (S. cerevisiae)], Genebank NM_002417 (Antigen identified by monoclonal antibody Ki-67), Gene registration Number (Genebank) N M_002776 (Kallikrein 10), gene registration number (Genebank) NM_002895 [Retinoblastoma-like 1 (p107)], gene registration number (Genebank) NM_003504 [CDC45 cell division cycle 45-like (S. cerevisiae)], gene registration number (Genebank) NM_003579 [RAD54-like (S. cerevisiae)], Genebank NM_003858 (Chromosome 14 open reading frame 65), Genebank NM_003981 (Protein regulator of cytokinesis 1), Genebank NM_004091 (E2F transcription factor 2), Gene Genebank NM_004523, Kinesin family member 11, Genebank NM_004526 [MCM2 minichromosome maintenance deficient 2, mitotin (S. cerevisiae)], Genebank NM_004701 (Cyclin B2), Gene Registration Number (Genebank) NM_005427 (Tumor protein p73), Genebank NM_005441 [Chromatin assembly factor 1, subunit B (p60)], Genebank (Genebank) NM_005504 (Branched chain aminotransferase 1, cytosolic), Gene registration number ( Genebank) NM_005915 [MCM6 minichromosome maintenance deficient 6 (MIS5 homolog, S. pombe) (S. cerevisiae)], Genebank NM_005938 [Myeloid / lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 7], Genebank NM_006101 (Kinetochore associated 2), Gene accession number (Genebank) NM_006306 [SMC1 structural maintenance of chromosomes 1-like 1 (yeast), Gene accession number (Genebank) NM_006739 [MCM5 minichromosome maintenance deficient 5, cell division cycle 46 (S. cerevisiae)], Genebank NM_006845 (Kinesin family member 2C), Genebank NM_01211 [(TPX2, microtubule-associated, homolog (Xenopus laevis)] , Gene Registration Number (Genebank) NM_012415 [RAD54 homolog B (S. cerevisiae)], Gene Registration Number (Genebank) NM_013975 (Ligase III, DNA, ATP-dependent), Gene Registration Number (Genebank) NM_014708 (Kinetochore associated 1), Gene Registration Number (Genebank) NM_014750 [Discs, large homolog 7 (Drosophila)], Gene Registration Number (Genebank) NM_015097 (Cytoplasmic linker associated protein 2), Gene Registration Number (Genebank) NM_015714 (G0 / G1switch 2), Gene Registration Number (Genebank) NM_015895 (Geminin, DNA repli cation inhibitor), Genebank NM_016169 (Suppressor of fused homolog (Drosophila)], Genebank NM_016272 (Transducer of ERBB2, 2), Genebank NM_016343 [Centromere protein F, 350 / 400ka (mitosin)], Genebank NM_016426 (G-2 and S-phase expressed 1), Genebank NM_018560 (WW domain containing oxidoreductase), Genebank NM_020238 (Inner centromere protein antigens 135 / 155kDa), genebank NG_024342 (Glucocorticoid receptor DNA binding factor 1), genebank NM_024680 (E2F transcription factor 8), genebank NM_031966 (Cyclin B1), gene registry Number (Genebank) NM_057749 (Cyclin E2), Gene Registration Number (Genebank) NM_138555 (Kinesin family member 23), Gene Registration Number (Genebank) NM_144665 (Sestrin 3), Gene Registration Number (Genebank) NM_152526 [Amyotrophic lateral sclerosis 2 (juvenile) chromosome regio n, candidate 19], Genebank NM_182776 [MCM7 minichromosome maintenance deficient 7 (S. cerevisiae)], Genebank NM_183380 (Dystonin), Genebank (Genebank) U65410 [MAD2 mitotic arrest deficient-like 1 (yeast)], Genebank XM_034274 [V-myb myeloblastosis viral oncogene homolog (avian)-like 1], gene registration number (Genebank) XM_496557 (Plasminogen-like B2), gene registration number (Genebank) NM_000189 (Hexokinase 2), gene registration number (Genebank) AF285167 [ATP-binding cassette, sub-family A (ABC1), member 1), Gene Registration Number (Genebank) BX648281 [Low density lipoprotein receptor (familial hypercholesterolemia)], Gene Registration Number (Genebank) NM_005165 (Aldolase C, fructose-bisphosphate), Gene Registration Number (Genebank) XM_113962 ( Structural maintenance of chromosomes flexible hinge domain containing 1), Genebank AF285167 [ATP-binding cassette, sub-family A (ABC1), member 1], Genebank X66610 (enolase alpha, lung-specific ), Gene Registration Number (Genebank) NM_022755 (Inositol 1, 3,4,5,6-pentakisphosphate 2-kinase), gene registration number (Genebank) NM_014465 (Sulfotransferase family, cytosolic, 1B, member 1), gene registration number (Genebank) NM_014762 (24-dehydrocholesterol reductase), gene registration number (Genebank) NM_000689 (Aldehyde dehydrogenase 1 family, member A1), Gene Registration Number (Genebank) NM_006389 (Hypoxia up-regulated 1), Gene Registration Number (Genebank) AL833741 [Enolase 1, (alpha)], Gene Registration Number (Genebank) NM_002541 [Oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide)], gene accession number (Genebank) BC000054 (7-dehydrocholesterol reductase), gene accession number (Genebank) AK223395 (Fructose-1,6-bisphosphatase 1), gene registration number (Genebank) NM_182470 (Pyruvate kinase, muscle), Gene Registration Number (Genebank) AB053172 (DNA replication factor), Gene Registration Number (Genebank) AF360549 (BRCA1 interacting protein C-terminal helicase 1), Gene Registration Number (Genebank) AJ007669 ( Fanconi anemia, complementation group G), gene Genebank AK093320 [Target of EGR1, member 1 (nuclear)], Gene accession number (Genebank) AK123010 (Ribonucleotide reductase M2 polypeptide), Gene accession number (Genebank) AY032677 [Polymerase (DNA directed), theta], Gene Genebank AY281068 (Polybromo 1), Gene Registration Number (Genebank) BC034480 (Bloom syndrome), Gene Registration Number (Genebank) BC038666 (Fanconi anemia, complementation group D2), Gene Registration Number (Genebank) BC067848 [Karyopherin alpha 2 (RAG cohort 1, importin alpha 1)], gene registration number (Genebank) BX537939 [Elastin (supravalvular aortic stenosis, Williams-Beuren syndrome)], gene registration number (Genebank) D80008 (DNA replication complex GINS protein PSF1), gene registration Number (Genebank) L08238 [Suppressor of variegation 3-9 homolog 1 (Drosophila)], gene registration number (Genebank) NM_000059 (Breast cancer 2, early onset), gene registration number (Genebank) NM_000946 (Primase, polypeptide 1, 49 kDa) , Gene Registration Number (Genebank) NM_0 01003689 [L (3) mbt-like 2 (Drosophila)], Genebank NM_001067 [Topoisomerase (DNA) II alpha 170kDa], Genebank NM_001254 [CDC6 cell division cycle 6 homolog (S. cerevisiae)], Genebank NM_001269 (Regulator of chromosome condensation 1), Gene Registration Number (Genebank) NM_002689 [Polymerase (DNA directed), alpha 2 (70kD subunit)], Genebank NM_002692 [Polymerase (DNA directed), epsilon 2 (p59 subunit)], Genebank NM_002915 [Replication factor C (activator 1) 3, 38 kDa], Genebank (Menebank) NM_002916 [Replication factor C (activator 1) 4, 37 kDa], gene registration number (Genebank) NM_003258 (Thymidine kinase 1, soluble), gene registration number (Genebank) NM_003362 (Uracil-DNA glycosylase), gene registration number (Genebank) NM_003621 [PTPRF interacting protein, binding protein 2 (liprin beta) 2)], Genebank NM_004111 (Flap structure-specific endonuclease 1), Genebank NM_004153 [Origin recognition complex, subunit 1-like (yeast)], Genebank NM_005441 [Chromatin assembly factor 1, subunit B (p60)], gene registration number (G enebank) NM_005914 [MCM4 minichromosome maintenance deficient 4 (S. cerevisiae)], gene registration number (Genebank) NM_006306 [SMC1 structural maintenance of chromosomes 1-like 1 (yeast)], gene registration number (Genebank) NM_006591 [Polymerase (DNA- directed), delta 3, accessory subunit], Genebank NM_006892 [DNA (cytosine-5-)-methyltransferase 3 beta], Genebank NM_012117 [Chromobox homolog 5 (HP1 alpha homolog, Drosophila)] , Genebank NM_012415 [RAD54 homolog B (S. cerevisiae)], Genebank NM_014321 [Origin recognition complex, subunit 6 homolog-like (yeast)], Genebank (Genebank) NM_015895 (Geminin, DNA replication inhibitor), Genebank NM_016937 [Polymerase (DNA directed), alpha].

1) Among the marker genes, marker genes whose expression is increased by treatment of cardiovascular toxicity-inducing drugs are as follows:

Genebank No. NM_078487 [cyclin-dependent kinase inhibitor 2b (p15, inhibits cdk4)], Genebank No. X06374 (platelet-derived growth factor alpha polypeptide), Genebank No. AK057315 [testis specific a2 homolog (mouse)], gene bank number AK127033 (dynamin 2), gene bank number (Genebank) AB209596 (g protein pathway suppressor 1), gene bank number (Genebank) AB209095 [cell division cycle 2-like 1 (pitslre) proteins)], gene accession number (Genebank) AF033122 (sestrin 1), gene registration number (Genebank) NM_053056 (cyclin d1), gene accession number (Genebank) AY123223 (Sestrin 2), gene accession number (Genebank) AK001361 [Protein phosphatase 1, regulatory (inhibitor) subunit 15A], Genebank NM_003550 [MAD1 mitotic arrest deficient-like 1 (yeast)], Genebank NM_139314 (Angiopoietin-like 4), Genebank (Genebank) NM_078467 [Cyclin-dependent kinase inhibitor 1A (p21, Cip1) ], Gene Registration Number (Genebank) NM_003311 (Pleckstrin homology-like domain, family A, member 2), Gene Registration Number (Genebank) AK125880 (Tumor protein p53 inducible nuclear protein 1), Gene Registration Number (Genebank) AB208907 (Macrophage erythroblast) attacher), Genebank AB096256 [Unc-5 homolog B (C. elegans)], Genebank AK092970 [protein (peptidylprolyl cis / trans isomerase) nima-interacting 1], Genebank CR623038 (inhibitor of dna binding 2, dominant negative helix-loop-helix protein), Genebank (Genebank) NM_033238 (promyelocytic leukemia), Genebank (Genebank) NM_003236 (transforming growth factor, alpha), Genebank (Genebank) BC060797 [par-6 partitioning defective 6 homolog gamma (c. Elegans)], Gene Registration Number (Genebank) NM_078467 [cyclin-dependent kinase inhibitor 1a (p21, cip1)], Gene Registration Number (Genebank) AY123223 (sestrin 2), Gene Registration Number (Genebank) NM_004642 (cdk2-associated protein 1), Gene Registration Genebank NM_033030 [bol, boule-like (drosophila)], Genebank NM_004359 (cell division cycle 34), Genebank CR612719 (growth arrest and dna-damage-inducible, alpha), Genebank BC064996 [rho guanine nucleotide excha nge factor (gef) 1], genebank NM_002838 (protein tyrosine phosphatase, receptor type, c), genebank NM_006455 (synaptonemal complex protein sc65), genebank AK074112 (cyclin l2) ), Genebank (Genebank) BM462208 (proliferating cell nuclear antigen), Genebank (Genebank) NM_02041 [poly (rc) binding protein 4], Genebank (Genebank) NM_006396 (sjogren's syndrome / scleroderma autoantigen 1), gene Registration number (Genebank) NM_003620 (protein phosphatase 1d magnesium-dependent, delta isoform), gene registration number (Genebank) CR590366 [midkine (neurite growth-promoting factor 2)], gene registration number (Genebank) NM_057158 (dual specificity phosphatase 4) ), Genebank AK001361 [protein phosphatase 1, regulatory (inhibitor) subunit 15a], Genebank (Genebank) NM_002419 (mitogen-activated protein kinase kinase kinase 11), Genebank (Menebank) NM_013258 (pyd and c ard domain containing), Genebank NM_013366 (anaphase promoting complex subunit 2), Genebank NM_012127 (cdkn1a interacting zinc finger protein 1), Genebank BC040303 (protein tyrosine phosphatase type iva, member 1), Genebank NM_002487 (necdin homolog (mouse)], Genebank (Genebank) NM_001759 (cyclin d2), Genebank (Genebank) NM_002632 (placental growth factor, vascular endothelial growth factor-related protein Genebank NM_005343 (v-ha-ras harvey rat sarcoma viral oncogene homolog), Genebank (Genebank) BC006795, M92424 [mdm2, transformed 3t3 cell double minute 2, p53 binding protein (mouse)], Genebank (Genebank) NM_003550 [mad1 mitotic arrest deficient-like 1 (yeast)], Genebank (Genebank) AK074652 (serta domain containing 1), Genebank (Genebank) BC094797 [unc-84 homolog b (c. elegans)], genebank (Genebank) CR612719 (growth arrest and dna-damage-inducible, alpha), genebank (Genebank) NM_181805 [protein kinase (camp-dependent, catalytic) inhibitor gamma], genebank (Genebank) ) M15169 (adrenergic, beta-2-, receptor, surface), Genebank AY247738 [tribbles homolog 3 (drosophila)], Genebank number AK074652 (serta domain containing 1), Gene registry number (Genebank) ) NM_139314 (angiopoietin-like 4), Gene Registration Number (Genebank) BX641114 (annexin a4), Gene Registration Number (Genebank) NM_002744 (protein kinase c, zeta), Gene Registration Number (Genebank) R612719 (growth arrest and dna-damage -inducible, alpha) Genebank BM557864 (heat shock 27kda protein 1), Genebank NM_001024807 [amyloid beta (a4) precursor-like protein 1], Genebank NM_003311 (pleckstrin) homology-like domain, family a, member 2), Genebank N M_020418 [poly (rc) binding protein 4], gene registration number (Genebank) AF332558 (bcl2 binding component 3), gene registration number (Genebank) NM_003811 [tumor necrosis factor (ligand) superfamily, member 9], gene registration number (Genebank) ) AK125880 (tumor protein p53 inducible nuclear protein 1), Genebank AK128462 (cisplatin resistance related protein crr9p), Genebank AK001361 [protein phosphatase 1, regulatory (inhibitor) subunit 15a], gene registration number (Genebank) AB208907 (macrophage erythroblast attacher), Genebank AB209361 [fas (tnf receptor superfamily, member 6)], Genebank NM_013258 (pyd and card domain containing), Genebank (Genebank) AB096256 [unc-5 homolog b (c. elegans), Genebank NM_004309 [rho gdp dissociation inhibitor (gdi) alpha], Genebank (Genebank) BC063507 (heat shock 70kda protein 1a), Genebank BG033621 (tumor protein, translationally- controlled 1), Genebank NM_003841 (tumor necrosis factor receptor superfamily, member 10c, decoy without an intracellular domain), Genebank NM_003806 [harakiri, bcl2 interacting protein (contains only bh3 domain)], gene Genebank CR611266 (fas-activated serine / threonine kinase), Gene Registration Number (Genebank) AK126489 (tubulin, beta), Gene Registration Number (Genebank) NM_003820 [tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry mediator) Genebank AK122614 (peroxisome proliferative activated receptor, delta).

2) Among the marker genes, marker gene genes whose expression is reduced by the treatment of cardiovascular toxicity-inducing drugs are as follows:

Gene Registration Number AB020641 [PFTAIRE protein kinase 1], Gene Registration Number AB033068 [Fizzy / cell division cycle 20 related 1 (Drosophila)], Gene Registration Number AB057597 (Testis-specific kinase 2), Gene Registration Number AB188493 (MAP / microtubule affinity regulating kinase 2), gene accession number AB208919 [Fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome)], gene accession number AB209154 (Receptor tyrosine kinase-like orphan receptor 2), gene accession number AB209179 [Polo -like kinase 1 (Drosophila)], gene accession number AB209337 [CDC7 cell division cycle 7 (S. cerevisiae)], gene accession number AB209615 (protein phosphatase 5, catalytic subunit), gene accession number AB209681 [Rap guanine nucleotide exchange factor ( GEF) 4], gene accession number AF041210 [Midline 1 (Opitz / BBB syndrome)], gene accession number AF045459 (BMX non-receptor tyrosine kinase), gene accession number AF053305 [BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) ], Gene registration number AF053306 [BUB1 budding uninhibited by benzimidazoles 1 homolog beta (yeast)], gene registration number AF332220 [Transducin (beta) -like 1Y-linked], gene registration number AF375884 (Protein O-fucosyltransferase 1), gene registration No. AK000581 [Unc-51-like kinase 4 (C. elegans)], gene accession number AK055221 (F-box protein 5), gene accession number AK056691 [Germ cell associated 2 (haspin)], gene accession number AK074377 (Chromosome 19 open reading frame 31), gene accession number AK091289 (Zinc finger protein 367), gene accession number AK092352 (Weakly similar to serine / threonine protein kinase Kp78), gene accession number AK123004 (Microtubule associated serine / threonine kinase-like), gene accession number AK131531 [Cyclin-dependent kinase (CDC2-like) 10 )], Gene registration number AL833316 [Membrane-associated ring finger (C3HC4) 8], gene registration number AY257469 [Citron (rho-interacting, serine / threonine kinase 21)], gene registration number BC011498 (Histone deacetylase 6), gene registration Number BC015416 (Ring finger protein 32), gene accession number BC032677 (Ubiquitin-conjugating enzyme E2C), gene accession number BC035407 [Interleukin 3 receptor, alpha (low affinity)], gene accession number BC035514 [TEK tyrosine kinase, endothelial (venous malforal) mations, multiple cutaneous and mucosal], gene registration number BC038295 (Mitogen-activated protein kinase kinase 7), gene registration number BC043502 [never in mitosis gene a-related kinase 2], gene registration number BC070168 (Tetraspanin 8) , Gene registration number BC071593 (V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog), gene registration number BC081541 [Poly (ADP-ribose) polymerase family, member 12], gene registration number BX537987 (Tripartite motif-containing 59), Gene registration number CR749621 (SNF related kinase), gene registration number CR933728 (Cell division cycle 2, G1 to S and G2 to M), gene registration number DQ097177 (HECT, UBA and WWE domain containing 1), gene registration number NM_000513 [Opsin 1 (cone pigments), medium-wave-sensitive (color blindness, deutan)], gene registration number NM_001005862 [V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro / glioblastoma derived oncogene homolog (avian)], gene registration number NM_001014796 (Discoidin domain recep tor family, member 2), gene registration number NM_001274 [CHK1 checkpoint homolog (S. pombe)], gene registration number NM_001490 [Glucosaminyl (N-acetyl) transferase 1, core 2 (beta-1,6-N-acetylglucosaminyltransferase)], gene registration number NM_001543 [N-deacetylase / N-sulfotransferase (heparan glucosaminyl) 1 ], Gene registration number NM_001618 [Poly (ADP-ribose) polymerase family, member 1], gene registration number NM_001789 (Cell division cycle 25A), gene registration number NM_001790 (Cell division cycle 25C), gene registration number NM_001798 (Cyclin-dependent kinase 2), gene registration number NM_002612 (Pyruvate dehydrogenase kinase, isozyme 4), gene registration number NM_002648 (Pim-1 oncogene), gene registration number NM_002758 (Mitogen-activated protein kinase kinase 6), gene registration number NM_002834 [Protein tyrosine phosphatase , non-receptor type 11 (Noonan syndrome 1)], gene accession number NM_002839 (Protein tyrosine phosphatase, receptor type, D), gene accession number NM_002843 (Protein tyrosine phosphatase, receptor type, J), gene accession number NM_002982 [Chemokine ( CC motif) ligand 2], gene registration number NM_003242 (Transforming growth factor, beta receptor II (70 / 80kDa)), gene registration number NM_003318 (TTK protein kinase), gene registration number NM_003335 (Ubiquitin-activating enzyme E1-like), gene Accession number NM_003384 (Vaccinia related kinase 1), gene accession number NM_003607 [CDC42 binding protein kinase alpha (DMPK-like)], gene accession number NM_004318 (Aspartate beta-hydroxylase), gene accession number NM_004667 (Hect domain and RLD 2), Gene registration number NM_004687 (Myotubularin related protein 4), gene registration number NM_004938 (Death-associated protein kinase 1), gene registration number NM_005082 (Tripartite motif-containing 25), gene registration number NM_005188 [Cas-Br-M (murine) ecotropic retroviral transforming sequence], gene registration number NM_005391 (Pyruvate dehydrogenase kinase, isozyme 3), gene registration number NM_005483 [Chromatin assembly factor 1, subunit A (p150)], gene registration number NM_005983 [S-phase kinase-asso ciated protein 2 (p45)], gene registration number NM_006904 (Protein kinase, DNA-activated, catalytic polypeptide), gene registration number NM_007039 (Protein tyrosine phosphatase, non-receptor type 21), gene registration number NM_007295 (Breast cancer 1, early onset), gene accession number NM_007313 (V-abl Abelson murine leukemia viral oncogene homolog 1), gene accession number NM_007375 (TAR DNA binding protein), gene accession number NM_014176 [Ubiquitin-conjugating enzyme E2T (putative)], gene accession number NM_014238 (Kinase suppressor of ras 1), gene accession number NM_014264 [Polo-like kinase 4 (Drosophila)], gene accession number NM_014501 (Ubiquitin-conjugating enzyme E2S), gene accession number NM_014502 [PRP19 / PSO4 pre-mRNA processing factor 19 homolog (S. cerevisiae)], gene registration number NM_014671 (Ubiquitin protein ligase E3C), gene registration number NM_014791 (Maternal embryonic leucine zipper kinase), gene registration number NM_015148 (PAS domain containing serine / threonine kinase), gene registration number NM_015967 [Protein tyrosine phosphatase, non-receptor type 22 (lymphoid)], gene registration number NM_016513 [Intestinal cell (MAK-like) kinase], gene registration number NM_016653 (Hypothetical protein LOC339751), gene registration number NM_020205 (Zinc finger, A20 domain containing 1), gene Accession number NM_020438 (Dolichyl pyrophosphate phosphatase 1), gene accession number NM_020914 (Chromosome 17 open reading frame 27), gene accession number NM_021643 [Tribbles homolog 2 (Drosophila)], gene accession number NM_022455 (Nuclear receptor binding SET domain protein 1), Gene Registration No. NM_033389 [Slingshot homolog 2 (Drosophila), Gene Registration No. NM_080836 (Serine / threonine kinase 35), Gene Registration No. NM_153498 (Calcium / cal modulin-dependent protein kinase ID), gene registration number NM_172207 (Calcium / calmodulin-dependent proteinkinase kinase 1, alpha), gene registration number NM_173216 (ST6nbeta-galactosamide alpha-2,6-sialyltranferase 1), gene registration number NM_198268 (Homeodomain interacting protein kinase 1), gene accession number U67156 (Mitogen-activated protein kinase kinase kinase 5), gene accession number (Genebank) AB033337 (M-phase phosphoprotein 1), gene accession number (Genebank) AB187578 [Shugoshin-like 1 (S. pombe), Genebank AB209271 (Ets2 repressor factor), Genebank (Genebank) AF345347 (Sperm associated antigen 5), Genebank (Jenebank) AJ421269 (Regulator of chromosome condensation 2), Gene registry number ( Genebank) AK091170 [Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4)], gene accession number (Genebank) AK096393 (DIRAS family, GTP-binding RAS-like 3), gene accession number (Genebank) AK096541 (M-phase phosphoprotein 9), Genebank AL713771 (Nuclear transcription factor Y, gamma), Genebank AL832666 [Establishment of cohesion 1 homolog 2 (S. cerevisiae)], Genebank AL833052 (Hypothetical protein FLJ40137), Genebank AL833191 [SMC2 structural maintenance of chromosomes 2-like 1 (yeast)], Genebank AY366508 (Loss of heterozygosity, 11, chromosomal region 2, gene A), Gene registry number (Genebank) AY367065 [Asp (abnormal spindle) ) -like, microcephaly associated (Drosophila)], Genebank BC004352 (Kinesin family member 22), Genebank BC011000 (Cell division cycle associated 5), Genebank BC033086 [Transcription factor 19 (SC1)], Genebank BC038666 (Fanconi anemia, complementation group D2), Genebank BC050369 (E2F transcription factor 1), Genebank BC073878 (Kinesin family member C1), Genebank BX640816 (Nibrin), Genebank BX641109 [Spindle assembly 6 homolog (C. elegans), Genebank No. CR592757 [Barren homolog 1 (Drosophila)], Genebank No. D79987 [Extra spindle poles like 1 (S. cerevisiae)], Genebank No. NM_000059 (Breast cancer) 2, early onset, Genebank NM_000189 (Hexokinase 2), Genebank NM_001005414 (ZW10 interactor antisense), Genebank NM_001012271 (Effector cell peptidase receptor 1), Gene registry number ( Genebank) NM_001254 [CDC6 cell division cycle 6 homolog (S. cerevisiae)], gene accession number (Genebank) NM_001761 (Cyclin F), gene accession number (Genebank) NM_001813 (Centromere protein E, 312kDa), gene registration number (Genebank) NM_001827 (CDC28 protein kinase regulatory subunit 2), Genebank NM_002388 [MCM3 minichromosome maintenance deficient 3 (S. cerevisiae)], Genebank NM_002417 (Antigen identified by monoclonal antibody Ki-67), Gene registration Number (Genebank) NM _002776 (Kallikrein 10), gene registration number (Genebank) NM_002895 [Retinoblastoma-like 1 (p107)], gene registration number (Genebank) NM_003504 [CDC45 cell division cycle 45-like (S. cerevisiae)], gene registration number (Genebank) NM_003579 [RAD54-like (S. cerevisiae)], Genebank NM_003858 (Chromosome 14 open reading frame 65), Genebank NM_003981 (Protein regulator of cytokinesis 1), Genebank NM_004091 (E2F transcription factor 2), Gene Genebank NM_004523, Kinesin family member 11, Genebank NM_004526 [MCM2 minichromosome maintenance deficient 2, mitotin (S. cerevisiae)], Genebank NM_004701 (Cyclin B2), Gene Registration Number (Genebank) NM_005427 (Tumor protein p73), Genebank NM_005441 [Chromatin assembly factor 1, subunit B (p60)], Genebank (Genebank) NM_005504 (Branched chain aminotransferase 1, cytosolic), Gene registration number ( Genebank) NM_005915 [MCM6 minichromosome maintenance deficient 6 (MIS5 homolog, S. pombe) (S. cerevisiae)], Genebank NM_005938 [Myeloid / lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 7], Genebank NM_006101 (Kinetochore associated 2), Gene accession number (Genebank) NM_006306 [SMC1 structural maintenance of chromosomes 1-like 1 (yeast), Gene accession number (Genebank) NM_006739 [MCM5 minichromosome maintenance deficient 5, cell division cycle 46 (S. cerevisiae)], Genebank NM_006845 (Kinesin family member 2C), Genebank NM_01211 [(TPX2, microtubule-associated, homolog (Xenopus laevis)] , Gene Registration Number (Genebank) NM_012415 [RAD54 homolog B (S. cerevisiae)], Gene Registration Number (Genebank) NM_013975 (Ligase III, DNA, ATP-dependent), Gene Registration Number (Genebank) NM_014708 (Kinetochore associated 1), Gene Registration Number (Genebank) NM_014750 [Discs, large homolog 7 (Drosophila)], Gene Registration Number (Genebank) NM_015097 (Cytoplasmic linker associated protein 2), Gene Registration Number (Genebank) NM_015714 (G0 / G1switch 2), Gene Registration Number (Genebank) NM_015895 (Geminin, DNA repli cation inhibitor), Genebank NM_016169 [Suppressor of fused homolog (Drosophila)], Genebank NM_016272 (Transducer of ERBB2, 2), Genebank NM_016343 [Centromere protein F, 350 / 400ka (mitosin)], Genebank NM_016426 (G-2 and S-phase expressed 1), Genebank NM_018560 (WW domain containing oxidoreductase), Genebank NM_020238 (Inner centromere protein antigens 135 / 155kDa), genebank NM_024342 (Glucocorticoid receptor DNA binding factor 1), genebank NM_024680 (E2F transcription factor 8), genebank NM_031966 (Cyclin B1), gene Genebank NM_057749 (Cyclin E2), Genebank NM_138555 (Kinesin family member 23), Genebank NM_144665 (Sestrin 3), Genebank NM_152526 [Amyotrophic lateral sclerosis 2 juvenile) chromosome region , candidate 19], Genebank NM_182776 [MCM7 minichromosome maintenance deficient 7 (S. cerevisiae)], Genebank NM_183380 (Dystonin), Genebank (Genebank) U65410 [MAD2 mitotic arrest deficient-like 1 (yeast)], Genebank XM_034274 [V-myb myeloblastosis viral oncogene homolog (avian) -like 1], gene registration number (Genebank) XM_496557 (Plasminogen-like B2), gene registration number (Genebank) NM_000189 (Hexokinase 2), gene registration number (Genebank) AF285167 [ATP-binding cassette, sub-family A (ABC1), member 1), Gene Registration Number (Genebank) BX648281 [Low density lipoprotein receptor (familial hypercholesterolemia)], Gene Registration Number (Genebank) NM_005165 (Aldolase C, fructose-bisphosphate), Gene Registration Number (Genebank) XM_113962 ( Structural maintenance of chromosomes flexible hinge domain containing 1), Genebank AF285167 [ATP-binding cassette, sub-family A (ABC1), member 1], Genebank X66610 (enolase alpha, lung-specific ), Gene Registration Number (Genebank) NM_022755 (Inositol 1,3 , 4,5,6-pentakisphosphate 2-kinase, Genebank NM_014465 (Sulfotransferase family, cytosolic, 1B, member 1), Genebank NM_014762 (24-dehydrocholesterol reductase), Gene accession number (Genebank) NM_000689 (Aldehyde dehydrogenase 1 family, member A1), Gene Registration Number (Genebank) NM_006389 (Hypoxia up-regulated 1), Gene Registration Number (Genebank) AL833741 [Enolase 1, (alpha)], Gene Registration Number (Genebank) NM_002541 [Oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide)], gene accession number (Genebank) BC000054 (7-dehydrocholesterol reductase), gene accession number (Genebank) AK223395 (Fructose-1,6-bisphosphatase 1), gene registration number (Genebank) NM_182470 (Pyruvate kinase, muscle), Gene Registration Number (Genebank) AB053172 (DNA replication factor), Gene Registration Number (Genebank) AF360549 (BRCA1 interacting protein C-terminal helicase 1), Gene Registration Number (Genebank) AJ007669 ( Fanconi anemia, complementation group G), gene Genebank AK093320 [Target of EGR1, member 1 (nuclear)], Gene accession number (Genebank) AK123010 (Ribonucleotide reductase M2 polypeptide), Gene accession number (Genebank) AY032677 [Polymerase (DNA directed), theta], Gene Genebank AY281068 (Polybromo 1), Gene Registration Number (Genebank) BC034480 (Bloom syndrome), Gene Registration Number (Genebank) BC038666 (Fanconi anemia, complementation group D2), Gene Registration Number (Genebank) BC067848 [Karyopherin alpha 2 (RAG cohort 1, importin alpha 1)], gene registration number (Genebank) BX537939 [Elastin (supravalvular aortic stenosis, Williams-Beuren syndrome)], gene registration number (Genebank) D80008 (DNA replication complex GINS protein PSF1), gene registration Number (Genebank) L08238 [Suppressor of variegation 3-9 homolog 1 (Drosophila)], Genebank NM_000059 (Breast cancer 2, early onset), Gene accession number (Genebank) NM_000946 (Primase, polypeptide 1, 49 kDa) ), Genebank NM_00 1003689 [L (3) mbt-like 2 (Drosophila)], Genebank NM_001067 [Topoisomerase II alpha 170 kDa], Genebank NM_001254 [CDC6 cell division cycle 6 homolog (S. cerevisiae)], Genebank NM_001269 (Regulator of chromosome condensation 1), Genebank NM_002689 [Polymerase (DNA directed), alpha 2 (70kD subunit)], Genebank NM_002692 [Polymerase (DNA directed), epsilon 2 (p59 subunit)], Genebank NM_002915 [Replication factor C (activator 1) 3, 38 kDa], Genebank NM_002916 [Replication factor C (activator 1) 4, 37kDa], Genebank NM_003258 (Thymidine kinase 1, soluble), Genebank NM_003362 (Uracil-DNA glycosylase), Genebank NM_003621 [PTPRF interacting protein, binding protein 2 (liprin beta) 2)], Genebank NM_004111 (Flap structure-specific endonuclease 1), Genebank NM_004153 [Origin recognition complex, subunit 1-like (yeast)], Genebank NM_005441 [Chromatin assembly factor 1, subunit B (p60)], gene registration number (Gen ebank) NM_005914 [MCM4 minichromosome maintenance deficient 4 (S. cerevisiae)], gene registration number (Genebank) NM_006306 [SMC1 structural maintenance of chromosomes 1-like 1 (yeast)], gene registration number (Genebank) NM_006591 [Polymerase (DNA) -directed), delta 3, accessory subunit], Genebank NM_006892 [DNA (cytosine-5-)-methyltransferase 3 beta], Gene accession number (Genebank) NM_012117 [Chromobox homolog 5 (HP1 alpha homolog, Drosophila) ], Genebank NM_012415 [RAD54 homolog B (S. cerevisiae)], Genebank NM_014321 [Origin recognition complex, subunit 6 homolog-like (yeast)], Genebank (Genebank) NM_015895 (Geminin, DNA replication inhibitor), Genebank NM_016937 [Polymerase (DNA directed), alpha].

In order to discover marker genes for cardiovascular toxicity-induced drug search, the present inventors treated cytokines with human umbilical vein endothelial cells (HUVECs) showing daunorubicin showing cardiovascular toxicity. As a result, the daunorubicin was confirmed to be toxic to human umbilical vascular endothelial cell line (see FIG. 1), and the concentration of daunorubicin was determined based on the experiment. Subsequently, Daunorubicin was treated to human umbilical vascular endothelial cell line at the determined concentration, mRNA was isolated from the drug-treated cell line, and synthesized with cDNA and labeled with Cy5 fluorescent material. Labeled as material. The fluorescently labeled cDNA was hybridized with a 44k human whole genome oligomicroarray chip (Agilent, USA), and the fluorescence images were scanned to analyze differences in gene expression patterns (see FIGS. 2 and 3). . In the analysis, when the Cy5 / Cy3 ratio was 2.0 times or more, it was classified as an expression increased gene. As a result, it was confirmed that the gene with increased expression was 0.87% (384 out of 444291 genes) and the 1.39% with reduced expression (615 out of 44,291 genes). At this time, when genes changed by 2.0 times or more of expression by daunorubicin are classified by function, apoptosis, cell cycle, DNA metabolism, and protein, which are judged to act on cardiovascular toxicity, are identified. Genes involved in the regulation of protein kinase activity, the regulation of transferase activity and alcohol metabolism were selected (see Table 2 and Table 3). These genes have not been reported to be associated with toxicity in human umbilical vascular endothelial cells when treated with daunorubicin used in the present invention.

When treated with daunorubicin, alcohol metabolism causes dysfunction by regulating myocardial contractile function (Alvaro Mordente., Et . al ,. Lubmb Life 83-88. 2001).

Daunorubicin has been reported to reduce the expression of specific genes associated with blood vessels. Genes with reduced expression have reported PAI-1 associated with blood vessels. Anthracycline antibiotic daunorubicin decreases PAI-1 (Plasminogen activator inhibitor-1) gene expression in HUVEC cells. PAI-1 inhibits the breakdown of fibrin, a major factor in atherosclerosis. In this gene mutation, the fibrosis accumulates in the blood vessels and causes clogging of the arteries to promote atherosclerosis. High levels of PAI-1 (Plasminogen activator inhibitor-1) in the body increase the risk of heart attack. Decreased gene expression of PAI-1 also increases the expression of TNF-a (Tumor necrosis factor) genes, leading to apoptosis (Shinji Soeda., et al ., Biochimica et Biophysica Acta 234-241, 2001).

Treatment with daunorubicin in chronic myelogenous leukemia cells showed increased Bcl-2 protein and increased phosphorylation of p53 protein by FLT3 near the membrane. Genes with reduced expression have been reported with apoptosis-related Mcl-1 and Hdm2.

Daunorubicin not only binds DNA or RNA to inhibit protein synthesis, but also acts on topoisomerase II to cut the DNA chain. It is a drug with various mechanisms of action that binds to cell membranes and affects the transport system or shows cytotoxicity by the formation of oxygen radicals (Line Wergelasdl.,et al ., BioMed Central 1-11. 2007).

Daunorubicin inhibits the protein kinase C pathway. It is reported that the production of daunorubicin resistance results in the leakage of modulator P-170 glycoprotein, topoisomerase II, and high expression of bcl-2. Be sure to administer carefully because it causes cell necrosis outside the blood vessels (Shiwei Duan., et al . , Cancer Res 1-9, 2007).

In addition, the present invention provides a DNA microarray chip for cardiovascular toxicity-induced drug search in which an oligonucleotide, or a complementary strand molecule thereof, containing all or part of the marker gene gene sequence is integrated.

The oligonucleotide or complementary strand molecule thereof comprises 18 to 30 nucleic acids of the marker gene, preferably 20 to 25 nucleic acids.

The DNA microarray chip for cardiovascular toxicity-inducing drug screening of the present invention can be manufactured by a method known to those skilled in the art. The method of fabricating the microarray chip is as follows:

In order to immobilize the detected marker gene as a probe DNA molecule on a substrate of a DNA chip, a micropipetting method or a pin-shaped spotter using a piezoelectric method is used. It is preferable to use the method used, but is not limited thereto. In a preferred embodiment of the present invention, a microarray, which is a pin-shaped spotter, is used. The substrate of the DNA microarray chip is preferably coated with one active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, It is not limited to this. In addition, the substrate may be any one selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane, and nitrocellulose membrane, but is not limited thereto. In a preferred embodiment of the present invention, amino-silane-coated slide glass was used.

In addition, the present invention provides a cardiovascular toxicity-inducing drug screening method using the bio-barker.

The present invention

1) treating the test compound to human umbilical vascular endothelial cells;

2) separating RNA from the experimental group cells treated with the test compound of step 1) and control cells not treated with the test compound;

3) synthesizing RNA of the experimental group and the control group of step 2) with cDNA and labeling the fluorescent substance of the experimental group and the control group, respectively;

4) hybridizing cDNA labeled with different fluorescent materials of step 3) with DNA microarray chips;

5) analyzing the reacted DNA microarray chip of step 4); And

6) provides a cardiovascular toxicity-inducing drug search method comprising the step of confirming the expression level of the marker gene of the present invention in the analyzed data of step 5) compared to the control.

In the search method, the human umbilical vascular endothelial cells of step 1) is preferably HUVEC, but is not limited thereto, and any human umbilical vascular endothelial cells and cells derived from the tissue can be used.

In the search method, the fluorescent material of step 3) is Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (rhodamine-B-isothiocyanate, RITC), rhodamine (rhodamine) is preferably any one selected from the group consisting of, but not limited to, any fluorescent material known to those skilled in the art can be used.

In the search method, the DNA microarray chip of step 5) preferably uses Whole Human Genome Oligo Microarray (Agilent, USA) and the like, but is not limited thereto. Any microarray chip loaded with a low expression gene (see Tables 2 and 3) can be used, and it is most preferable to use a DNA microarray chip produced by the present inventors. In addition, the analysis method of step 5) preferably uses GenePix 4.1 software (Axon Instruments, USA), but is not limited thereto. Any analysis software known to those skilled in the art may be used.

In addition, the present invention

1) treating the test compound to human umbilical vascular endothelial cells;

2) separating RNA from the test cell treated with the test compound of step 1) and the control cell not treated with the test compound;

3) performing a real-time reverse transcript polymerase chain reaction (RT-PCR) using a primer complementary to the marker gene of the present invention and amplifying the marker gene of the RNA of step 2); And

4) provides a cardiovascular toxicity-inducing drug search method comprising the step of confirming the expression level by comparing the gene product of step 3) with the control.

In the search method, the human umbilical vascular endothelial cells of step 1) is preferably HUVEC, but is not limited thereto, and any human umbilical vascular endothelial cells and cells derived from the tissue can be used.

In the above search method, the primer of step 3) is complementary to the marker gene gene searched in the present invention, can amplify the marker gene, and any primers designed to be 100 to 300 bp (base pair) amplification products are used. It is possible.

In addition, the present invention provides a kit for cardiovascular toxicity-inducing drug search comprising the DNA microarray chip.

The DNA microarray chip is preferably used by the method of the present invention, but is not limited thereto.

The present invention provides a kit for detecting and diagnosing daunorubicin, which further comprises human umbilical cord vascular endothelial cells. The human umbilical vascular endothelial cells are preferably HUVEC, but are not limited thereto. Any human umbilical vascular endothelial cells may be used as long as they are derived from human umbilical vascular endothelial cells and tissues.

In addition to the search kit may include a fluorescent material, the fluorescent material is a streptadin-like phosphatease conjugate (strepavidin-like phosphatease conjugate), chemiflurorensce and chemiluminescent (chemiluminescent) Preferably, but not limited to selected from the group consisting of, in the preferred embodiment of the present invention used Cy3 and Cy5.

The detection kit may further include a reaction reagent, which is a buffer solution used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTP (premixed or separated feed), and fluorescent dyes. It may be composed of a labeling reagent such as a chemical inducing agent, a washing buffer, and the like, but is not limited thereto. The reaction reagents required for hybridization of DNA microarray chips known to those skilled in the art may be included.

In addition to the search kit may include a primer that is complementary to the marker gene gene, and can amplify the marker gene gene, the primer is complementary to the marker gene, can amplify the marker gene gene and 100 amplification products Both forward or reverse primer pairs designed to be from 300bp can be used.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Cell Culture and Chemical Treatment

<1-1> Cell Culture

HUVEC cells (CC-2517, CAMBREX, USA), a human umbilical vascular endothelial cell line, were 100 mm using EBM-2 medium (CAMBREX, USA) supplemented with 2% FBS and growth factors (CC-4176, CAMBREX, USA). The dish was incubated to 5 × 10 ⁵ cells / ml. The present inventors selected daunorubicin, which is a representative drug showing cardiovascular toxicity as a side effect, and dissolved in DMSO through existing studies and reports. Vehicle concentration was less than 0.1% in all experiments.

<1-2> MTT assay and chemical treatment

MTT experiments using HUVEC cell lines were performed by the method of Mossman et al . ( J. Immunol . Methods , 65, 55-63, 1983). Cells were treated with daunorubicin dissolved in DMSO in EBM-2 medium (CAMBREX, USA) at 10 × 10 ⁴ / well cell number in 24-well plates and after 48 hours, MTT (3- (4,5-dimethylthiazol- 5 mg / ml of 2,5-diphenyltetra zolium bromide) was added to the tube and incubated for 3 hours at 37 ° C. After that, the medium was removed and the formed formazan crystal was dissolved in 500 µl of DMSO. The plate was transferred to a -well plate, aliquoted, and the OD value was measured at an absorbance of 540 nm. The cytotoxicity of daunorubicin in the HUVEC cell line showed that the 20% survival inhibition concentration (IC ₂₀ ) was 191.00 ng / ml (Fig. 1), microarray experiments were performed by determining the concentrations.

Example 2 Microarray Experiment

<2-1> Isolation of Target RNA and Fluorescent Labeling

After dispensing HUVEC cells in a 100 mm dish at a concentration of 1.65 × 10 ⁶ cells / ml, the concentration of daunorubicin determined in Example 1-2 was treated for 48 hours. Thereafter, the whole cells were separated from the treated cells using a trizol reagent (Invitrogen life technologies, USA) according to the manufacturer's method, and purified using the RNeasy mini kit (Qiagen, USA). Genomic DNA was removed using RNase-free DNase set (Qiagen, USA) during RNA purification. The amount of each total RNA sample was measured by spectrophotometer, and the purity was confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies, USA).

<2-2> Labeled cDNA Preparation

CDNA was prepared using the total RNA of the daunorubicin treated group obtained in Example <2-1> for oligo microarray analysis. The total RNA obtained above was mixed with 30 μg of oligo (dT) primer and 2 μg (1 μg / μl), reacted at 65 ° C. for 10 minutes, and then annealed in ice. Reagents were mixed as shown in Table 1 for Reverse Transcript reaction of the annealed RNA.

Configuration Volume (μl) 5X first strand buffer 6 dNTPs 0.6 0.1 M DDT 3 SuperScript II Enzyme 3 Cy-3 or Cy-5 dUTP 2

Total RNA isolated from control HUVEC cell line was labeled with Cy3-dUTP (green), and RNA isolated from daunorubicin treated HUVEC cell line was labeled with Cy5-dUTP (red). At this time, the two samples were mixed and purified using a Microcon YM-30 column (Millipore, USA).

<2-3> hybridization reaction

Hybridization and washing procedures were carried out according to the instructions of Genochem Co., Ltd. Hybridization was performed in a 62 ° C. oven for 12 hours. A 44 k Whole Human Genome oligo microarray (Agilent, USA) was used as the DNA microarray chip. After washing (washing in 2 × SSC / 0.1% SDS for 2 minutes, washing in 1 × SSC for 3 minutes, 0.2 × SSC for 2 minutes), the slides were dried by centrifugation at 800 rpm for 3 minutes.

<2-4> Fluorescence Image Acquisition

Hybridization images on the slides were scanned with the Genepix 4000B (Axon Instruments, USA). Chips washed with unbound genes were obtained with a fluorescence image using a laser fluorescence scanner. In this case, the green fluorescence image in the control group, the red fluorescence image represents the activity level of the gene specifically expressed only in the experimental group, and the yellow fluorescence image is the complementary color of green and red, which means that there is no significant difference between the two groups. Scanned images were analyzed with GenePix 4.1 software (Axon Instruments, USA) to obtain gene expression rates. Marker genes for daunorubicin were selected from the data thus obtained (see FIG. 2).

As a result, 0.87% (384 of 44,291 genes) expressed genes showing an increase in the expression of Cy5 / Cy3 by 2.0 times or more by daunorubicin among the approximately 44,000 genes present on the oligo chip. This reduced gene was found to be 1.39% (615 out of 44,291 genes).

At this time, when genes changed by 2.0 times or more of expression by daunorubicin are classified by function, apoptosis, cell cycle, DNA metabolism, and protein, which are judged to act on cardiovascular toxicity, are identified. Genes involved in the regulation of protein kinase activity, the regulation of transferase activity and alcohol metabolism were selected (see Table 2 and Table 3). The genes have not been reported to be associated with toxicity in human umbilical vascular endothelial cells when treated with daunorubicin used in the present invention.

To daunorubicin  Genes with increased expression Registration Number (Genebank) Gene abbreviation Gene name Ratio of medians (1) cell cycle AK057315 TSGA2 testis specific a2 homolog (mouse) 2.05309 AF033122 SESN1 sestrin 1 2.79039 NM_053056 CCND1 cyclin d1 2.14236 NM_003236 TGFA transforming growth factor, alpha 2.24130 NM_033238 PML promyelocytic leukemia 3.65163 AY123223 SESN2 sestrin 2 2.63663 NM_006455 SC65 synaptonemal complex protein sc65 2.70626 NM_020418 PCBP4 poly (rc) binding protein 4 2.01122 NM_006396 SSSCA1 sjogren's syndrome / scleroderma autoantigen 1 2.19973 NM_003620 PPM1D protein phosphatase 1d magnesium-dependent, delta isoform 2.28086 AK001361 PPP1R15A protein phosphatase 1, regulatory (inhibitor) subunit 15a 2.01752 NM_057158 DUSP4 dual specificity phosphatase 4 2.79719 NM_012127 CIZ1 cdkn1a interacting zinc finger protein 1 2.55424 NM_001759 CCND2 cyclin d2 2.44304 NM_002632 PGF placental growth factor, vascular endothelial growth factor-related protein 2.22896 NM_003550 MAD1L1 mad1 mitotic arrest deficient-like 1 (yeast) 2.07140 (2) regulation of protein kinase activity NM_078487 CDKN2B cyclin-dependent kinase inhibitor 2b (p15, inhibits cdk4) 2.02176 AB209596 GPS1 g protein pathway suppressor 1 2.24663 NM_181805 PKIG protein kinase (camp-dependent, catalytic) inhibitor gamma 2.95804 NM_013366 ANAPC2 anaphase promoting complex subunit 2 2.92252 (3) apoptosis AK127033 DNM2 dynamin 2   3.29723 NM_139314 ANGPTL4 angiopoietin-like 4   5.79447 AB209095 CDC2L2 cell division cycle 2-like 1 (pitslre
proteins)   2.55619 NM_078467 CDKN1A cyclin-dependent kinase inhibitor 1a (p21, cip1)   8.10811 NM_001024807 APLP1 amyloid beta (a4) precursor-like protein 1   6.35267 NM_003311 PHLDA2 pleckstrin homology-like domain, family a, member 2   2.21963 AK125880 TP53INP1 tumor protein p53 inducible nuclear protein 1   2.15787 AB208907 MAEA macrophage erythroblast attacher   2.03114 AB096256 UNC5B unc-5 homolog b (c. elegans)   2.11226 NM_003841 TNFRSF10C tumor necrosis factor receptor superfamily,
member 10c, decoy without an intracellular domain   2.63799 NM_003806 HRK harakiri, bcl2 interacting protein (contains only bh3 domain)   3.05562 NM_003820 TNFRSF1 tumor necrosis factor receptor superfamily,
member 14 (herpesvirus entry mediator)   2.16602 (4) regulation of transferase activity NM_002419 MAP3K11 mitogen-activated protein kinase kinase
kinase 11 3.33089 NM_002838 PTPRC protein tyrosine phosphatase, receptor type, c 2.54511 AY247738 TRIB3 tribbles homolog 3 (drosophila) 2.31361 AK074652 SERTAD1 serta domain containing 1 2.03357

To daunorubicin  Genes whose expression is reduced Registration Number (Genebank) Gene abbreviation Gene name Ratio of medians (1) cell cycle AB033068 FZR1 Fizzy / cell division cycle 20 related 1
(Drosophila) 0.42943 AB033337 MPHOSPH1 M-phase phosphoprotein 1 0.32913 AB187578 SGOL1 Shugoshin-like 1 (S. pombe) 0.23275 AB209179 PLK1 Polo-like kinase 1 (Drosophila) 0.07319 AB209271 ERF Ets2 repressor factor 0.3478 AF053305 BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) 0.06571 AF053306 BUB1B BUB1 budding uninhibited by benzimidazoles 1 homolog beta (yeast) 0.05547 AF345347 SPAG5 Sperm associated antigen 5 0.07229 AJ421269 TD-60 Regulator of chromosome condensation 2 0.44479 AK056691 GSG2 Germ cell associated 2 (haspin) 0.32192 AK074377 UHRF1 Chromosome 19 open reading frame 31 0.07671 AK091170 CDKN2C Cyclin-dependent kinase inhibitor 2C (p18,
inhibits CDK4) 0.12231 AK096393 ARHI DIRAS family, GTP-binding RAS-like 3 0.41212 AK096541 MPHOSPH9 M-phase phosphoprotein 9 0.40989 AK131531 CDK10 Cyclin-dependent kinase (CDC2-like) 10 0.44758 AL713771 NFYC Nuclear transcription factor Y, gamma 0.48498 AL832666 ESCO2 Establishment of cohesion 1 homolog 2 (S.
cerevisiae) 0.43779 AL833052 FLJ40137 Hypothetical protein FLJ40137 0.4844 AL833191 SMC2L1 SMC2 structural maintenance of chromosomes 2-like 1 (yeast) 0.21063 AY257469 CIT Citron (rho-interacting, serine / threonine
kinase 21) 0.10936 AY366508 LOH11CR2A Loss of heterozygosity, 11, chromosomal
region 2, gene A 0.32248 AY367065 ASPM Asp (abnormal spindle) -like, microcephaly
associated (Drosophila) 0.03951 BC004352 KIF22 Kinesin family member 22 0.17767 BC011000 CDCA5 Cell division cycle associated 5 0.0695 BC032677 UBE2C Ubiquitin-conjugating enzyme E2C 0.13309 BC033086 TCF19 Transcription factor 19 (SC1) 0.07014 BC043502 NEK2 NIMA (never in mitosis gene a) -related kinase 2 0.04214 BC050369 E2F1 E2F transcription factor 1 0.28667 BC073878 KIFC1 Kinesin family member C1 0.24991 BX640816 NBS1 Nibrin 0.48659 BX641109 DKFZp761A078 Spindle assembly 6 homolog (C. elegans) 0.32725 CR592757 BRRN1 Barren homolog 1 (Drosophila) 0.07665 CR933728 CDC2 Cell division cycle 2, G1 to S and G2 to M 0.03382 D79987 ESPL1 Extra spindle poles like 1 (S. cerevisiae) 0.10605 NM_000189 HK2 Hexokinase 2 0.22601 NM_001005414 ZWINT ZW10 interactor antisense 0.14056 NM_001012271 BIRC5 Effector cell peptidase receptor 1 0.09013 NM_001761 CCNF Cyclin F 0.37686 NM_001789 CDC25A Cell division cycle 25A 0.26275 NM_001790 CDC25C Cell division cycle 25C 0.04715 NM_001813 CENPE Centromere protein E, 312kDa 0.11374 NM_001827 CKS2 CDC28 protein kinase regulatory subunit 2 0.42546 NM_002417 MKI67 Antigen identified by monoclonal antibody
Ki-67 0.01213 NM_002758 MAP2K6 Mitogen-activated protein kinase kinase 6 0.30599 NM_002776 KLK10 Kallikrein 10 0.38859 NM_002895 RBL1 Retinoblastoma-like 1 (p107) 0.44428 NM_003318 TTK TTK protein kinase 0.03428 NM_003579 RAD54L RAD54-like (S. cerevisiae) 0.14413 NM_003858 CCNK Chromosome 14 open reading frame 65 0.48783 NM_003981 PRC1 Protein regulator of cytokinesis 1 0.07085 NM_004091 E2F2 E2F transcription factor 2 0.31906 NM_004523 KIF11 Kinesin family member 11 0.23936 NM_004526 MCM2 MCM2 minichromosome maintenance deficient 2, mitotin (S. cerevisiae) 0.14575 NM_004701 CCNB2 Cyclin B2 0.03772 NM_005427 TP73 Tumor protein p73 0.47574 NM_005483 CHAF1A Chromatin assembly factor 1, subunit A (p150) 0.36944 NM_005504 BCAT1 Branched chain aminotransferase 1, cytosolic 0.42509 NM_005915 MCM6 MCM6 minichromosome maintenance deficient 6 (MIS5 homolog, S. pombe) (S. cerevisiae) 0.27146 NM_005938 MLLT7 Myeloid / lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 7 0.3934 NM_005983 SKP2 S-phase kinase-associated protein 2 (p45) 0.48021 NM_006101 KNTC2 Kinetochore associated 2 0.03983 NM_006739 MCM5 MCM5 minichromosome maintenance deficient 5, cell division cycle 46 (S. cerevisiae) 0.28432 NM_006845 KIF2C Kinesin family member 2C 0.03695 NM_007295 BRCA1 Breast cancer 1, early onset 0.44938 NM_007313 ABL1 V-abl Abelson murine leukemia viral oncogene homolog 1 0.18529 NM_007375 TARDBP TAR DNA binding protein 0.42362 NM_012112 TPX2 TPX2, microtubule-associated, homolog (Xenopus laevis) 0.04405 NM_013975 LIG3 Ligase III, DNA, ATP-dependent 0.27978 NM_014264 PLK4 Polo-like kinase 4 (Drosophila) 0.06002 NM_014708 KNTC1 Kinetochore associated 1 0.38732 NM_014750 DLG7 Discs, large homolog 7 (Drosophila) 0.04628 NM_015097 CLASP2 Cytoplasmic linker associated protein 2 0.49449 NM_015714 G0S2 G0 / G1switch 2 0.44837 NM_016169 SUFU Suppressor of fused homolog (Drosophila) 0.39425 NM_016272 TOB2 Transducer of ERBB2, 2 0.40084 NM_016343 CENPF Centromere protein F, 350 / 400ka (mitosin) 0.01651 NM_016426 GTSE1 G-2 and S-phase expressed 1 0.16086 NM_016653 ZAK Hypothetical protein LOC339751 0.40223 NM_018560 WWOX WW domain containing oxidoreductase 0.41397 NM_020238 INCENP Inner centromere protein antigens 135 / 155kDa 0.42731 NM_024342 GRLF1 Glucocorticoid receptor DNA binding factor 1 0.27529 NM_024680 FLJ23311 E2F transcription factor 8 0.36544 NM_031966 CCNB1 Cyclin B1 0.04859 NM_057749 CCNE2 Cyclin E2 0.38421 NM_138555 KIF23 Kinesin family member 23 0.0728 NM_144665 SESN3 Sestrin 3 0.37186 NM_152526 ALS2CR19 Amyotrophic lateral sclerosis 2 (juvenile)
chromosome region, candidate 19 0.47827 NM_183380 DST Dystonin 0.44214 U65410 MAD2L1 MAD2 mitotic arrest deficient-like 1 (yeast) 0.07002 XM_034274 MYBL1 V-myb myeloblastosis viral oncogene homolog (avian) -like 1 0.28284 XM_496557 RGP1 Plasminogen-like B2 0.42893 (2) alcohol methabolism NM_002612 PDK4 pyruvate dehydrogenase kinase, isozyme 4 0.35557 NM_005391 PDK3 pyruvate dehydrogenase kinase, isozyme 3 0.37266 NM_000189 HK2 hexokinase 2 0.22600 AF285167 ABCA1 cholesterol efflux regulatory protein 0.41932 BX648281 LDLR low density lipoprotein receptor (familial
hypercholesterolemia) 0.37942 NM_002130 HMGCS1 3-hydroxy-3-methylglutaryl-coenzyme a
synthase 1 (soluble) 0.48082 NM_005165 ALDOC aldolase c, fructose-bisphosphate 0.49372 XM_113962 KIAA0650 structural maintenance of chromosomes
flexible hinge domain containing 1 0.42359 AF285167 ABCA1 atp-binding cassette, sub-family a (abc1), member 1 0.41932 X66610 ENO1B; HLE1 enolase alpha, lung-specific 0.33868 NM_022755 C9orf12 inositol 1,3,4,5,6-pentakisphosphate 2-kinase 0.44723 AF375884 POFUT1 protein o-fucosyltransferase 1 0.33105 NM_014465 SULT1B1 sulfotransferase family, cytosolic, 1b,
member 1 0.34589 NM_014762 DHCR24 24-dehydrocholesterol reductase 0.25654 NM_000689 NM_000689 aldehyde dehydrogenase 1 family, member a1 0.25165 NM_006389 HYOU1 hypoxia up-regulated 1 0.49384 AL833741 ENO1 enolase 1, (alpha) 0.34051 NM_002541 OGDH oxoglutarate (alpha-ketoglutarate)
dehydrogenase (lipoamide) 0.23662 BC000054 DHCR7 7-dehydrocholesterol reductase 0.45278 AK223395 FBP1 fructose-1,6-bisphosphatase 1 0.41066 NM_182470 PKM2 pyruvate kinase, muscle 0.46000 (3) DNA methabolism AB028450 NCOR1 Nuclear receptor co-repressor 1 0.29648 AB053172 CDT1 DNA replication factor 0.30334 AB209337 CDC7 CDC7 cell division cycle 7 (S. cerevisiae) 0.44491 AF095289 PTTG3 Pituitary tumor-transforming 3 0.17649 AF360549 BRIP1 BRCA1 interacting protein C-terminal helicase 1 0.30262 AJ007669 FANCG Fanconi anemia, complementation group G 0.37762 AK074377 UHRF1 Chromosome 19 open reading frame 31 0.07671 AK093320 TOE1 Target of EGR1, member 1 (nuclear) 0.47256 AK123010 RRM2 Ribonucleotide reductase M2 polypeptide 0.06679 AY032677 POLQ Polymerase (DNA directed), theta 0.0915 AY281068 PB1 Polybromo 1 0.46175 BC011498 HDAC6 Histone deacetylase 6 0.44705 BC034480 BLM Bloom syndrome 0.19473 BC038666 FANCD2 Fanconi anemia, complementation group D2 0.48627 BC067848 KPNA2 Karyopherin alpha 2 (RAG cohort 1, importin alpha 1) 0.32905 BX537939 ELN Elastin (supravalvular aortic stenosis,
Williams-Beuren syndrome) 0.48189 D80008 KIAA0186 DNA replication complex GINS protein PSF1 0.48368 DQ097177 UREB1 HECT, UBA and WWE domain containing 1 0.15108 L08238 SUV39H1 Suppressor of variegation 3-9 homolog 1
(Drosophila) 0.18829 NM_000059 BRCA2 Breast cancer 2, early onset 0.33063 NM_000946 PRIM1 Primase, polypeptide 1, 49kDa 0.28839 NM_001003689 L3MBTL2 L (3) mbt-like 2 (Drosophila) 0.48748 NM_001067 TOP2A Topoisomerase (DNA) II alpha 170kDa 0.01856 NM_001254 CDC6 CDC6 cell division cycle 6 homolog (S.
cerevisiae) 0.20776 NM_001269 CHC1 Regulator of chromosome condensation 1 0.49675 NM_001274 CHEK1 CHK1 checkpoint homolog (S. pombe) 0.40987 NM_001798 CDK2 Cyclin-dependent kinase 2 0.4747 NM_002388 MCM3 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) 0.41603 NM_002689 POLA2 Polymerase (DNA directed), alpha 2 (70kD subunit) 0.45176 NM_002692 POLE2 Polymerase (DNA directed), epsilon 2 (p59
subunit) 0.25833 NM_002915 RFC3 Replication factor C (activator 1) 3, 38 kDa 0.22105 NM_002916 RFC4 Replication factor C (activator 1) 4, 37 kDa 0.42826 NM_003258 TK1 Thymidine kinase 1, soluble 0.05323 NM_003362 UNG Uracil-DNA glycosylase 0.32924 NM_003504 CDC45L CDC45 cell division cycle 45-like (S.
cerevisiae) 0.07699 NM_003621 PPFIBP2 PTPRF interacting protein, binding protein 2 (liprin beta 2) 0.30163 NM_004111 FEN1 Flap structure-specific endonuclease 1 0.19996 NM_004153 ORC1L Origin recognition complex, subunit 1-like (yeast) 0.44115 NM_005441 CHAF1B Chromatin assembly factor 1, subunit B (p60) 0.34329 NM_005914 MCM4 MCM4 minichromosome maintenance deficient 4 (S. cerevisiae) 0.35446 NM_006306 SMC1L1 SMC1 structural maintenance of chromosomes 1-like 1 (yeast) 0.33196 NM_006591 POLD3 Polymerase (DNA-directed), delta 3, accessory subunit 0.43002 NM_006892 DNMT3B DNA (cytosine-5-)-methyltransferase 3 beta 0.3838 NM_006904 PRKDC Protein kinase, DNA-activated, catalytic
polypeptide 0.25843 NM_012117 CBX5 Chromobox homolog 5 (HP1 alpha homolog,
Drosophila) 0.42306 NM_012415 RAD54B RAD54 homolog B (S. cerevisiae) 0.29873 NM_014321 ORC6L Origin recognition complex, subunit 6
homolog-like (yeast) 0.48654 NM_015895 GMNN Geminin, DNA replication inhibitor 0.41952 NM_016937 POLA Polymerase (DNA directed), alpha 0.37117 NM_022455 NSD1 Nuclear receptor binding SET domain protein 1 0.29251 NM_022836 DCLRE1B DNA cross-link repair 1B (PSO2 homolog, S.
cerevisiae) 0.42494 NM_130398 EXO1 Exonuclease 1 0.11793 NM_144678 TOM1L2 Target of myb1-like 2 (chicken) 0.23585 NM_181578 RFC5 Replication factor C (activator 1) 5, 36.5 kDa 0.49855 NM_182776 MCM7 MCM7 minichromosome maintenance deficient 7 (S. cerevisiae) 0.22656 U47742 MYST3 MYST histone acetyltransferase (monocytic
leukemia) 3 0.46944 U81234 CXCL6 Chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) 0.30656

1 is a graph of cytotoxicity of human umbilical vascular endothelial cell line of daunorubicin,

2 and 3 show the results of analyzing the gene expression patterns of human umbilical vascular endothelial cell line treated with daunorubicin using a microarray chip.

Claims (15)

Apoptosis for treatment of daunorubicin in which all of the nucleic acid sequences of any one or more genes selected from the following or fragments of the above genes consisting of 18 to 30 nucleic acid sequences are integrated ) DNA microarray chip to check related gene expression: Genebank AK127033 (dynamin 2), Genebank AB209095 [cell division cycle 2-like 1 (pitslre proteins)], Genebank NM_003311 (Pleckstrin homology-like domain, family A, member 2), Genebank AK125880 (Tumor protein p53 inducible nuclear protein 1), Genebank AB096256 [Unc-5 homolog B (C. elegans)], Genebank NM_003841 (tumor necrosis factor receptor superfamily, member 10c, decoy without an intracellular domain, genebank NM_003806 [harakiri, bcl2 interacting protein (contains only bh3 domain)], genebank NM_003820 [tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry mediator)]. delete delete delete delete delete delete delete delete delete Claim 1 kit for confirming apoptosis-related gene expression for the treatment of daunorubicin comprising the DNA microarray chip. 12. The kit of claim 11, further comprising Human Umbilical Vein Endothelial Cells (HUVEC). The method of claim 11, further comprising any one selected from the group of fluorescent substances consisting of streptadin-like phosphatease conjugate, chemilurorensce and chemiluminescent. Kit characterized in that. The method according to claim 11, which is selected from the group of reaction reagents consisting of a buffer solution used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTP (premixed or separated feed), a marker reagent, and a wash buffer. Kit comprising any one further. delete

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