KR20080083865A - Compositions, dna chips for diagnosis of cancer confirming livin over-expression and pharmaceutical composition for treating cancer - Google Patents

Abstract

A composition for identifying expression of livin is provided to diagnose tumor inducing over-expression of the livin and be useful for treating the tumor by controlling the expression of the livin. A composition or a DNA chip for diagnosing tumor confirming over-expression of livin comprises a livin gene or a fragment including a consecutive sequence of 25-100 bp derived from the livin gene, wherein the tumor is infant leukemia. A composition for diagnosing the tumor comprises an antibody against a livin protein. A pharmaceutical composition for treating the tumor comprises livin as an effective ingredient. Further, the infant leukemia is an acute lymphoblastic leukemia.

Description

Composition, DNA Chips for Diagnosis of Cancer Confirming Livin Over-Expression and Pharmaceutical Composition for Treating Cancer

1 shows the expression level of ribin according to risk factors and chromosomal abnormalities. The maximum and minimum values were expressed in bars, and statistical analysis was performed using the Mann-Whitney U test.

Figure 2 shows the apoptosis response to chemotherapy (chemotherapeutic agent) according to the expression of the ribin [A: degree of expression of ribin when showing a poor response to the bone marrow response 7 days after drug administration; B: survival of cells following livin expression; C: Degree of expression of cytotoxic assay caspase 3 and poly (ADP-ribose) polymerase (PARP) proteins].

Figure 3 shows the survival without relapse according to the expression of ribin [A: showing the relationship between the expression and survival without recurrence; B: showing the relationship between ribin and relapse-free survival in high-risk patients.

Field of invention

The present invention relates to a diagnostic composition, a diagnostic DNA chip, and a tumor therapeutic pharmaceutical composition for tumors to confirm the overexpression of livin, and more specifically, to confirm whether or not the expression of ribin in a biological sample, such as pediatric acute lymphocytic leukemia The present invention relates to a tumor diagnostic composition for diagnosing tumors induced by overexpression, a diagnostic DNA chip, and a pharmaceutical composition for treating tumors that regulate overexpression of ribin.

Background

Acute lymphoblastic leukemia is the most common malignant tumor in children, accounting for a quarter of childhood tumors and about 75% of childhood leukemias. Diagnosis of childhood acute lymphocytic leukemia can be confirmed by prognostic factors such as the number, age, immunophenotype, and cytogenetic abnormality of the initial leukocytes (Smith, M. et al. , J. Clin. Oncol ., 14:18, 1996; Greaves, MF et al., Br. J. Haematol ., 48: 179, 1981; Pui, CH et al., N. Engl. J. Med ., 354: 166, 2006; Pui, CH et al., N. Engl. J. Med ., 350: 1535, 2004).

Recently, molecular biological studies on tumors have begun to develop new diagnostic modalities for diagnosis and treatment (Ramaswamy, S. et al., J. Clin. Oncol ., 20: 1932, 2002; Golub, TR et al. ., Curr. Opin.Hematol ., 8: 252, 2001; Ebert, BL et. al ., Blood , 104: 923, 2004). One of them is real-time RT-PCR to identify genes specifically expressed in acute lymphocytic leukemia.

Apoptosis is a biological mechanism that causes programmed cell death. When apoptosis does not occur, various diseases such as cancer appear. When apoptosis is stopped, cancer development and progression occur (Reed, CJ et al., Semin Hematol ., 37: 9, 2000). Thus, tumors can be treated by modulating anti-apoptosis proteins (Tamm, I. et. al ., Clin . Cancer . Res . 6: 1796, 2000; Wrzesien-Kus, A. et al ., Apoptosis , 9: 705, 2004; Herr, I. et al ., Blood , 98: 2603, 2001; Kawasaki, H. et al ., Cancer Res ., 58: 5071, 1998; Kawasaki, H. et al ., Cancer , 91: 2026, 2001). Over the last few decades, research has been conducted on complex networks of pro-apoptosis and anti-apoptosis proteins that regulate apoptosis (Yang, YL et al. al ., Cell Res ., 10: 169, 2000; Deveraux, QL et al ., Genes Dev ., 13: 239, 1999; Nachmias, B. et al ., Semin . Cancer Biol ., 14: 231, 2004; Zhang, HG et al ., Oncogene , 23: 2009, 2004). Expression of pro-apoptotic and anti-apoptotic proteins in malignant leukemia is known to vary according to the type of leukemia and the characteristics of each patient (Nakagawa, Y. et. al ., Leuk . Res ., 28: 487, 2004; Carter, BZ et al ., Blood , 102: 4179, 2003; Yamamoto, K. et al ., Leuk . Res ., 28: 1203, 2004; Wuchter, C. et al ., Haematologica. , 89: 363, 2004; Campos, L. et al ., Leukemia 10: 434, 1996). This difference is important in predicting response to treatment (Coustan-Smith, E. et. al ., Blood , 87: 1140, 1996; Hogarth, LA et al ., Blood , 93: 2671, 1999; Prokop, A. et al ., Leukemia , 14: 1606, 2000).

Recently, a group of proteins known as an inhibitor of apoptosis protein (IAP) has been identified. The group of IAP proteins is known to inhibit apoptosis induced by various stimuli (Nachmias, B. et. al ., Semin . Cancer Biol ., 14: 231, 2004; Schimmer, AD et al ., Cancer Res ., 64: 7183, 2004), which act as initiators and effectors of caspases.

One of the IAP protein families, livin (MIM 605737; baculoviral IAP repeat-containing 7, BIRC7; aliases, ML-IAP, or KIAP) was first discovered in melanoma cells and overexpressed in MCF-7 cells. Suicide was found to be inhibited. It was originally named ML-IAP because it was found in skin cancer cells, but it is increasingly written as ribin. Livin has a COOH-terminal RING finger domain and a baculoviral IAP repeat (BIR) domain, which is expected to exhibit anti-apoptotic acitivity.

Livin is located in the long arm of chromosome 20 on 20q13.3, the site of amplification in melanoma or other malignancy, and two splitting variants (α-isoform and β-isoform) are known ( Vucic, D. et al ., Curr . Biol ., 10: 1359, 2000), inhibits caspase 3,7,9 or inhibits suicide receptors and mitochondrial-based apoptosis pathways via JNK1 (c-Jun N-termianl kinase1) (Vucic, D. et. al ., Curr . Biol ., 10: 1359, 2000; Kasof, GM et. al ., J. Biol . Chem ., 276: 3238, 2001; Lin, JH et al ., Biochem . Biophys . Res . Commun ., 279: 820, 2000).

To date, only limited research has been conducted on the function of ribine as a diagnostic marker for malignant tumors, and the expression of ribine is still controversial in other types of malignant tumors. For example, ribin is expressed in superficial bladder cancer, melanoma, neuroblastoma, and the like, and is used as a diagnostic marker, but is not related to survival of patients with nasopharyngeal cancer. It is known to have been known (Gazzangia, P. et. al ., Ann . Oncol ., 14: 85, 2003; Nachmias, B, et al ., Cancer Res ., 63: 6340, 2003; Kim, DK et al ., Pediatr . Dev . Pathol ., 8: 621, 2005; Xiang, Y. et al ., Laryngoscope , 116: 126, 2006). However, the relationship between childhood acute lymphocytic leukemia and ribin expression has not been studied.

Accordingly, the present inventors have made efforts to develop a diagnostic composition, a diagnostic DNA chip, and a tumor therapeutic pharmaceutical composition for tumors that induce overexpression of ribine. As a result, ribine is specifically overexpressed in pediatric acute lymphocytic leukemia. The present invention was completed by confirming that it acts as a good prognostic factor of.

It is a main object of the present invention to provide a diagnostic composition for tumors containing a livin gene or a fragment having a continuous base sequence of 25 to 100 bp derived from the gene and confirming the overexpression of livin.

It is another object of the present invention to provide a composition for diagnosing a tumor containing an antibody to a ribin protein and confirming overexpression of ribin.

Still another object of the present invention is to provide a DNA chip for diagnosis of a tumor containing a ribin gene or a fragment thereof and confirming the overexpression of ribin.

Still another object of the present invention is to provide a pharmaceutical composition for treating tumor, which contains ribin as an active ingredient and controls overexpression of ribine.

In order to achieve the above object, the present invention provides a diagnostic composition for tumors containing a livin gene or a fragment having a continuous nucleotide sequence of 25 ~ 100bp derived from the gene, and confirms whether or not Libin overexpression.

The present invention also provides a composition for diagnosing tumors containing an antibody to the ribin protein and confirming whether or not the ribon is overexpressed.

The present invention also provides a diagnostic DNA chip for a tumor containing a ribin gene or a fragment having a nucleotide sequence of 25 to 100 bp derived from the gene and confirming whether overexpression of ribin is present.

The present invention also provides a pharmaceutical composition for treating tumor, which contains ribin as an active ingredient and controls overexpression of ribine.

In the present invention, the tumor may be characterized as pediatric leukemia, and the pediatric leukemia may be characterized as pediatric acute lymphoblastic leukemia, and the diagnosis is a diagnosis for the prognosis after the tumor treatment It can be characterized by.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a diagnostic composition for a tumor containing a ribin gene or the gene fragment and confirming the overexpression of ribin.

In the present invention, general prognostic factors (gender, age, leukocyte count, risk factor, immunophenotype, chromosome count, structural abnormalities of chromosomes, CSF involvement and mediastinal involvement, etc.) and treatment results such as bone marrow response and recurrence rate at 7 days after drug treatment The difference in the expression rate of livin was analyzed using Pearson chi-square test, and the difference in the expression level of ribin according to prognostic factors was analyzed using Mann-Whitney U test. Survival was performed using the Mann-Whitney U test. Overall survival rate (OS), relapse-free survival rate (RFS) and event-free survival rate (EFS) were measured using the Kaplan-Meier method.

According to the present invention, after synthesizing cDNA of mononuclear cells from bone marrow of pediatric acute lymphocytic leukemia patients, mRNA expression of ribin was measured by real-time RT-PCR using the cDNA as a template.

As a result, female patients, 1-9 years old, leukocyte count <50 × 10 ⁹ / L, patients with standard-risk, t (12; 21), cerebrospinal fluid (CSF) involvement or mediastinal mass ( media in patients with mediasinal mass, patients with hypodiplodynes less than 44 or patients with 45-49, patients with favorable prognostic factor and patients with good bone marrow response 7 days after drug administration. The expression rate was high. Therefore, it was possible to confirm whether acute lymphocytic leukemia in children by checking the expression of ribine. In addition, the expression of ribin was high in patients with good prognostic factors, and in patients with good bone marrow response at 7 days after drug administration, the level of ribin expression was high. Therefore, ribine is a good prognostic factor for childhood acute lymphocytic leukemia. I could see that.

In the present invention, only an example of a method for measuring the expression level of ribin by real-time RT-PCR, but there is no limitation as long as it is a method that can determine whether the expression of ribin. In other words, after electrophoresis of RNA in pediatric acute lymphocytic leukemia patients, the degree of expression of ribin was measured by Northern blot, or hybridized with a radioisotope labeled ribin DNA or RNA, or RT-PCR, In situ RNA hybridization, RNA-DNA hybridization ELISA, cDNA microarray, oligo microarray, etc. can be used.

In addition, in the present invention, only the ribin gene of SEQ ID NO: 1 is used, but it will be apparent to those skilled in the art that the present invention is not limited thereto.

In the present invention, the ribon gene fragment is preferably a nucleic acid fragment having a length capable of specifically binding to the ribin gene, more preferably a fragment having a continuous base sequence of 25 ~ 200bp derived from the ribin gene. In the present invention, even though only 25 bp ribin gene fragment can be used to specifically detect ribin, three 25 bp sequences were used. The 25 bp ribin gene fragment was 5'-TGTGGCCCCCTCCGTCCCTGCCTCT, and when it was blasted using only one base sequence, it showed a very specific degree as expect 0.00001. However, in the present invention, as well as the 25 bp ribin gene fragment, two more sequences were used, so the specificity would be higher, and considering the gap of each nucleotide sequence, a 100 bp ribin gene fragment may be used. Can be. In other words, the gene fragment was used to accurately and specifically detect the ribin gene.

Another aspect of the present invention relates to a diagnostic composition for a tumor containing an antibody to the ribin protein, and confirms whether or not the ribon overexpression.

According to the present invention, after treating the mononuclear cells isolated from the bone marrow of pediatric acute lymphocytic leukemia patients with methylprednisolone to induce apoptosis, the whole protein of the cells is extracted and electrophoresed, followed by anti-ribin monoclonal Antibodies were treated to determine the expression of ribin protein. As a result, expression of full-length ribin proteins (α- and β-isoforms) and cleaved forms of ribine remained constant.

Pediatric acute lymphocytic leukemia diagnostic composition according to the present invention is a method for overexpressing the ribin gene in the body by using a recombinant vector containing the ribin gene, a method for inducing the overexpression of the ribin gene in the body using a virus, by synthesizing a ribin protein oral It may be administered using a method of administration or infusion.

Another aspect of the present invention relates to a DNA chip for diagnosis of a tumor containing a ribin gene or a fragment thereof and confirming the overexpression of ribin.

The probe of the present invention is used as a gene chip. A preferred embodiment of the present invention provides a DNA chip in which the nucleic acid probe of the present invention is immobilized on a solid support. A DNA chip is a high density accumulation of fragments of several nucleotide sequences on a small substrate, which is used to detect unknown DNA by hybridization between DNA immobilized on the substrate and an unknown DNA sample complementary thereto. The advantage is that more than one gene can be searched quickly. In addition, in the case of Southern blot or Northern blot, a nitrocellulose membrane is used as a medium for attaching the dielectric material, whereas a DNA chip can attach a very small amount of dielectric material at a high density by using a solid agent such as glass. The advantage is that you can search for a large number of genes at the same time. DNA chips are classified into cDNA chips and oligonucleotide chips according to the size of the attached genetic material. At least 500bp of gene (full-length open reading frame, EST) is fixed to the cDNA chip, and oligonucleotide consisting of about 15 to 25 bases is fixed to the oligonucleotide chip.

The substrate on which the probe is to be fixed is made of inorganic materials such as glass and silicone, or polymer materials such as acrylic, polyethylene terephtalate (PET), polystylene, polycarbonate, polypropylene, and the like. The surface of may be used flat or having a plurality of holes (hole). The probe is fixed either covalently to the substrate at either 5 'or 3'. The immobilization method is a conventional technique, for example, using an electrostatic force or by attaching an amine group on the synthesized oligo to an aldehyde coated slide to induce bonding between the two, or on an amine group coated slide or L-lysine. It can be done on this coated slide or by incorporating a nitrocellulose membrane on the coated slide. As an aspect of the present invention, when synthesizing the probe, a base having an amino residue in the 3 'position is inserted to covalently bond to a glass plate coated with an aldehyde residue.

Fixing and arranging different probes on a substrate surface uses methods such as pin microarrays, inkjets, photolithography, electrical arrays, and the like. In one embodiment of the present invention using a microarrayer prepared according to a known method in a state in which the probe is dissolved in a buffer solution (Yoon et al ., J. Microbiol . Biotechnol ., 10 (1): 21, 2000). The principle of microarrays is that the microfabricated pins carry DNA from the plate and transfer it to the same location specified by the computer on the substrate. Between the amine groups on the 3 'position of the probe and the aldehyde groups coated on the glass plate, under conditions where the humidity is maintained between 45% and 65%, preferably between 50% and 55%, for fixing the probe transferred by the microarray. In order to facilitate the reaction, the reaction is induced for at least 1 hour, and left for at least 6 hours to fix the DNA probe.

If necessary to detect cells derived from a living organism or the organism itself, these cells may be partially or completely lysed by chemical and / or physical methods to facilitate access to the RNA and / or DNA of those cells and Contact. The contact can be carried out on a suitable support such as nitrocellulose, cellulose or nylon filters in a liquid medium or solution. Such contact can occur under optimum conditions, suboptimal conditions or limit conditions, which conditions include the temperature, the reactant concentration, the presence of substances that lower the base pair optimal temperature of the nucleic acid (eg formamide, dimethylsulfoxide and urea) and the reaction volume. And / or the presence of a substance (eg, dextran sulfate, polyethyleneglycol or phenol) to reduce and / or accelerate hybrid formation. Fluorescent materials used to analyze the hybridization results of DNA chips can be used Cy3-dUTP, Cy5-dUTP, Cy3-dCTP, Cy5-dCTP (Amersham Pharmacia, U.K.).

Another aspect of the present invention relates to a pharmaceutical composition for treating tumor, which contains ribin as an active ingredient and controls overexpression of ribine.

The pharmaceutical composition of the present invention can be formulated or used in combination with drugs such as antihistamines, anti-inflammatory drugs, anticancer agents and antibiotics that are already in use.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The pharmaceutical compositions according to the invention can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods. Can be. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

In particular, the present invention only pediatric acute lymphocytic leukemia as a tumor for inducing overexpression of ribine, tumors that are overexpressed when the tumor occurs, that is, melanoma, non small cell lung cancer (non small cell lung cancer, Crnkovic-Mertens) I. et al ,. Lung Cancer , 54 (2): 135, 2006), bladder cancer, Gazzaniga P. et al., Annual of onocology , 14 (1): 85, 2003), Neuroblastoma, Kim DK et al ., Pediatr . Dev . It will be apparent to those skilled in the art that the present invention can be applied to Pathol ., 8 (6): 621, 2005).

Example  1. Characteristics of Pediatric Acute Lymphoblastic Leukemia Patients

At the initial diagnosis, samples of 222 children with acute lymphoblastic leukemia under 15 years were collected at Samsung Medical Center and Seoul National University Hospital. The diagnosis of pediatric acute lymphocytic leukemia was determined by morphological changes of bone marrow cells by Wringht-Giemsa staining and immunophenotyping analyses of leukemic cells by flow cytometry. t (9; 22), t (12; 21), and 11q23 rearrangement chromosomal aberrations were determined by conventional cytogenetic analysis, including fluorescent in-situ hybridization (FISH).

In the sample of pediatric acute lymphocytic leukemia patients, the leukocyte count was 50 × 10 ⁹ / L, and the patients aged 1-9 years at the time of diagnosis were divided into the standard-risk group, and other patients were at high risk ( high-risk) group. Treatment protocols for standard-risk patients are described in the Pediatric Cancer Group CCG-1891 protocol (Lange, BJ et al ., Blood , 99: 825, 2002) or CCG-1952 protocol (Broxson, EH et. al ., Pediatr . Blood Cancer , 44: 226, 2005). CCG-1882 protocol (Nachman, J. et al., J. Clin. Oncol ., 16: 920, 1998) if the bone marrow leukemic blast at day 7 after induction chemotherapy is at least 25% . Was used by modifying. If the leukocytes are more than 25% at 14 days after induction chemotherapy, CCG-1882 protocol, CCG-106B protocol (Gaynon PS et al ., J. Clin. Oncol ., 11: 2234, 1993) ) Or the CCG-1901 protocol (Heath JA et al ., J. Clin. Oncol ., 21: 1612, 2003) was used.

The characteristics of the pediatric acute lymphocytic leukemia patients are shown in Table 1. The mean age of 222 patients (138 males, 84 females) was 65.5 months (range 3 to 190), the average leukocyte count was 9.00 × 10 ⁹ / L (range 0.31 to 925.1), and the average bone marrow leukocytes were 92.0. % (Range 31-100). 84.2% of patients with leukocytes> 75%, 129 standard-risk patients, 93 high-risk patients, and 171 patients (77.7%) exhibited the precursor B-cell immunophenotype. It was. There were 26 (12.8%) and 9 (4.4%) patients with hyperdiploidy 50 or higher and hypodiploidy 44 or lower, respectively, and for translocation confirmed by chromosome analysis and / or FISH, t (12; 21) There were 35 patients, 32 patients with t (9; 22) or 11q23 rearrangements, and 155 patients with no chromosomal abnormalities or with other chromosomal abnormalities.

Characteristics and Expression Rate of Ribin in Children with Acute Lymphoblastic Leukemia Number of patients Ribin expression P value Number Expression rate (%) gender Woman 84 28 33.3 man 138 29 21.0 .042 Age (years) 1-9 161 49 30.4 10 years old or older or under 1 year old 61 8 13.1 .008 White blood cell count <50 × 10 ⁹ / L 166 49 29.5 ≥50 × 10 ⁹ / L 56 8 14.3 .024 Risk group Standard-risk patient 129 42 32.6 High risk patients 93 15 16.1 .006 Immunophenotype Precursor B-cell 171 45 26.3 Etc 49 12 24.5 .797 Chromosome number 50 or more 26 One 3.8 45-49 168 48 28.6 44 or less 9 3 33.3 .023 Structural abnormalities of chromosomes t (12; 21) 35 29 82.9 Absence or other 155 27 17.4 <.001 t (9; 22) or 11q23 rearrangement 32 One 3.1 Whether CSF is involved absence 193 49 25.4 existence 12 One 8.3 .182 Mediastinal involvement absence 215 57 26.5 existence 7 0 0 .114 7 day BM response to treatment Leukocytes <25% 143 50 35.0 Leukocyte blasts ≥25% 69 6 8.7 <.001 Recurrence radish 185 56 30.3 U 37 One 2.7 <.001

Example  2. Pediatric Acute Lymphoblastic Leukemia In the patient Livin's  Expression rate and degree of expression

Mononuclear cells (MNCs) were isolated from 2 ml of the patient's bone marrow by ficoll density gradient centrifugation, and then total RNA was purified using a QIAamp RNA Blood kit (Qiagen, Hilden, Germany). Extracted. After DNA-free (Ambion, Austin, TX) treatment to remove chromosomal DNA, cDNA was synthesized using an oligo (dT) 15-mer primer by SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, Calif.).

Primer and livin gene (Hs00223384_m1, GeneBank accession number NM_139317, Applied Biosystems, SEQ ID NO: 1) provided as a template using the synthesized cDNA as a template and provided by ABI PRISM 7000 Sequence Detector System (Applied Biosystems, Foster City, CA) TaqMan GAPDH Control Reagents for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene combined with Assays-on-demand Gene Expression Set (Applied Biosystems) and TaqMan Universal PCR Master Mix (Applied Biosystems) Real-time RT-PCR was performed using (Applied Biosystems), followed by analysis using ABI PRISM 7000 Sequence Detection software (Applied Biosystems). The conditions of real-time RT-PCR were performed a total of 40 times of amplification for 2 minutes at 50 ° C, 10 minutes at 95 ° C, 15 seconds at 95 ° C, and 1 minute at 60 ° C.

The expression level of the ribin gene was determined by the ΔΔCt method (ΔΔCt = (Ctlivin-CtGAPDH) sample- (Ctlivin-CtGAPDH) calibrator) with an average ΔCt (Ctlivin-CtGAPDH) of 1.0 from all samples bound after the reaction. Analyzed.

As a result, ribin mRNA was expressed in 57 of 222 patients (25.7%), and the expression rate of ribin was male patients, younger than 1 year old or older than 10 years, leukocyte count below 50 × 10 ⁹ / L, high-risk ) Patients and patients with structural chromosomal abnormalities other than t (12; 21), female patients ( P = .042), 1 to 9 years old ( P = .008), leukocyte count 50 × 10 ⁹ / L ( P = .024), higher in standard-risk patients ( P = .006) and t (12; 21) chromosomal structural abnormalities ( P <.001). In addition, the expression rate of ribin was higher in patients without CSF involvement or mediastinal mass and with chromosome hypodiplody ≥ 50 than in patients with cerebral spinal fluid (CSF) involvement or mediasinal mass. It was higher in patients with hypodiplody ≤44 or in patients with 45-49 ( P = .023). There was no difference in the expression rate of livin according to immunophenotype (Table 1).

In patients with livin expression, the average level of expression of livin was 3.28 (range, 0.04 to 382.68), and patients with a favorable prognostic factor had higher expression than those with unfavorable prognostic factor. The degree was high.

As a result of measuring the expression of ribin according to the risk level, the expression level of ribin was higher in standard-risk patients (mean 0.68; range 0.04 ~ 382.68) than in high-risk patients (mean 0.68; range 0.04 ~ 11.71; P = .021). (FIG. 1A).

In addition, as a result of measuring the expression level of ribin according to the chromosomal abnormality, patients whose t (12; 21) (average 7.38; range 0.86 ~ 382.68) did not show t (12; 21) (average 1.26; range 0.04 ~) 37.75; P = .027) (FIG. 1B).

Example  3. Cytotoxicity Assay

Treatment of leukocyte blasts with methylprednisolone (methylprednisolone) 50 ㎍ / ㎖ for 24, 48, 72 hours, and then using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) Trazolium salt Cell proliferation was confirmed by measuring the degree of conversion of trazolium salt WST-8 to forazan (Kaspers, GJ et al., Blood , 90: 2723, 1997; Kaspers, GJ et al). , Blood , 92: 259, 1998).

In other words, leukocytes (1.5 × 10 ⁵ cells / well) of pediatric acute lymphocytic leukemia patients cultured in RPMI1640 medium containing 10% FBS (fetal bovine serum) and penicillin were transferred to 96-well plates. After treatment with μg / ml, the cells were incubated at 37 ° C for 24, 48 and 72 hours, and then treated with 10 μl of WST-8 (Dojindo Lab, Kumamoto, Japan), respectively, to convert WST-8 into four majors. Absorbance was measured by incubating for 4 hours at. Cell number was measured by hemocytometer by treatment with trypan blue (final concentration 0.2%) for 5 minutes.

The expression rate of ribin was unfavorable in the bone marrow response at 7 days after drug administration (unfavorable day 7 bone marrow response (leukocyte ≧ 25%), compared to patients at 7 days after drug administration. A favorable day 7 bone marrow response, leukocytes <25%) was higher (35.0% vs. 8.7%, P <.001). In addition, the proportion of patients with poor results was lower in patients with ribin expression (10.7% vs. 40.4%, P <.001, A in FIG. 2) than patients without ribin expression.

As a result of measuring the survival rate of leukocytes induced by apoptosis by treating methylprednisolone as described above, the survival rate of leukocytes was low when ribin was expressed (FIG. 2B).

Monocytic cells isolated from the patient's bone marrow were treated with lysis buffer (0.8% ammonium chloride solution, StemCell Technology, Vancouver, Canada) and washed three times with PBS (phosphate-buffered saline). , RPMI1640 medium containing 10% FBS (fetal bovine serum) and penicillin was incubated to a final concentration of 1 × 10 ⁷ / ㎖ was treated with 50 ㎍ / ㎖ methyl prednisone. After culturing the cells for 0, 24, 48, 72 hours, the cells were collected and the whole protein was extracted with Pro-Prep solution (Intron, Seoul, Korea), and then in 10% Bis-Tris pre-cast gel (Invitrogen). After development, transfer to a polyvinylidene difluoride membrane (Millipore, Bedford, Mass.), And then dissolve the membrane in 5% skim milk in TBST (Tris-buffered saline) containing 0.1% Tween 20. Block at room temperature for hours.

Afterwards, anti-ribin monoclonal antibodies (clone 88C570, Abcam, Cambridge, UK), anti-caspase 3 antibodies (Cell Signaling Technology, Beverly, MA) and antipoly (ADP-ribose) polymerase (poly (ADP-ribose) polymerase , PARP) antibody (Cell Signaling Technology) diluted 1: 1000, and anti-β-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) 1: 5000 diluted to the membrane overnight at 4 ℃ After washing 3 times with TBST buffer for 5 minutes, and then treated with anti-rabbit or anti-mouse IgG (Cell Signaling Technology) and reacted at room temperature for 1 hour to confirm with ECL detection kit (Amersham Bioscience, Piscataway, NJ). It was.

Expression of full-length livin proteins (α- and β-isoforms) and cleaved forms of livin are described by Nachmias, B. et. al ., Cancer Res . Confirmed by the method described in 63: 6340, 2003, specific ribin protein and cleaved protein fragments could be observed using anti-ribin monoclonal antibodies (clone 88C570, ABcam, Cambridge, UK). The amount of ribin protein was measured by GS-800 image densitometer (Bio-Rad, Taiwan), and the expression levels of ribin and β-tubulin were measured using Quantity on 2 4.2.2 software (Bio-Rad, CA). The ratio of tubulin to livin was defined as the degree of ribin protein expression.

As a result, expression of full-length ribin proteins (α- and β-isoforms) and cleaved forms of ribins remained constant, and caspase 3 and poly (ADP-ribose) polymerases The expression of (ADP-ribose) polymerase (PARP) protein increased as apoptosis progressed (FIG. 2C).

Example  4. Livin  Relationship between manifestations and recurrence

The pediatric acute lymphocytic leukemia patients were divided into the Libin expression group and the Libin non-expression group, and the survival rate of each group was compared using the log-rank test (SPSS 10.0), and the event was defined as recurrence rate or treatment delayed death. Univariate analysis and multivariate analysis were performed by using Cox regression analysis (SPSS 10.0) compared to general prognostic factors for relapse free survival or event free survival. The relationship between the degree of ribin mRNA expression and protein expression was measured using the Spearman test (SPSS 10.0).

The mean duration of survival of 193 patients among the 222 patients was 37 months (range 1-90), 37 patients recurred and 7 died of delayed treatment. The 5 year old OS, RFS, and EFS (± 95% confidence interval, CI) of the 222 patients were 82.2 ± 7.0%, 78.6 ± 6.8% and 76.0 ± 6.8%, respectively (Table 2).

Investigation of the relationship between expression and recurrence of ribin showed higher relapse-free survival (RFS) in patients without ribin expression (97.9 ± 4.0% vs. 64.9 ± 11.8%, P <.001, A of FIG. 3.

All patients with high-risk expression did not relapse (FIG. 3B), and among 29 patients transplanted with stem cells, the 5-year relapse-free survival (RFS) of 24 patients without livin expression was 50.0 ± 22.5. While 5% (P = .067), ribin expressed five patients survived without relapse.

According to univariate analyses, in addition to leukocyte counts of less than 50 × 10 ⁹ / L, precursor B-cell immunophenotypes, and t (12; 21) chromosomal abnormalities, the expression of ribin may serve as a good prognostic factor. And t (9; 22) or 11q23 rearrangement acted as an unfavorable prognostic factor. According to multivariate analyses, the expression of ribin, structural abnormalities and age of chromosomes were independent of good prognostic factors of pediatric acute lymphocytic leukemia, and leukocyte counts, immunophenotypes and chromosome numbers were associated with prognostic factors.

Unilateral and Plural Analysis of Prognostic Factors for Survival Without Recurrence Number Recurrence Rate (%) 5 year RFS (± 95% CI) Unitary analysis Pluralistic analysis HR 95% CI P value HR 95% CI P value gender Woman 84 9 (10.7) 87.4 ± 7.7 man 138 28 (20.3) 73.9 ± 9.3 1.864 0.879-3.950 .104 1.367 0.599-3.120 .458 Age (years) 1-9 161 20 (12.4) 83.4 ± 7.3 ≥10 or <1 61 17 (27.8) 65.3 ± 14.2 3.425 1.778 to 6.598 <.001 2.095 0.996-4.406 .051 White blood cell count <50 × 10 ⁹ / L 166 20 (12.0) 83.6 ± 8.9 ≥50 × 10 ⁹ / L 56 17 (30.4) 61.7 ± 15.2 3.457 1.806 -6.617 <.001 1.537 0.617 ~ 3.827 .356 Immune phenotype Precursor B-cell 171 24 (14.0) 80.8 ± 7.67 Etc 49 13 (26.5) 70.5 ± 14.2 2.139 1.088-4.204 .027 1.186 0.491 ~ 2.868 .705 Chromosome number .805 .425 50≥ 26 5 (19.2) 65.4 ± 29.0 45-49 168 27 (16.1) 81.6 ± 6.6 0.730 0.280-1.899 .518 0.499 0.171-1.455 .203 ≤44 9 2 (22.2) 75.0 ± 30.0 0.839 0.162 to 4.352 .834 0.689 0.118-4.038 .680 Chromosome structural abnormalities <.001 .033 t (12; 21) 35 2 (5.7) 93.6 ± 8.6 Absence or other 155 19 (12.3) 83.1 ± 7.7 2.342 0.545-10.054 .252 0.630 0.131-3.036 .565 t (9; 22) or 11q23 rearrangement 32 16 (50.0) 39.7 ± 23.0 12.742 2.923-55.550 .001 1.923 0.344 ~ 10.754 .457 Livin expression Expression 57 1 (1.8) 97.9 ± 4.0 Non-expression 165 36 (21.8) 71.4 ± 9.0 15.098 2.069 ~ 110.195 .007 8.844 1.010-77.479 .049

As described in detail above, the present invention has an effect of providing a diagnostic composition, a diagnostic DNA chip, and a pharmaceutical composition for treating tumors to confirm the overexpression of ribin. According to the present invention, since ribine is overexpressed in pediatric acute lymphocytic leukemia patients, it is possible to diagnose acute leukocytic leukemia by confirming the expression of ribine, and overexpression of ribine by administering a pharmaceutical composition containing ribine as an active ingredient. It is useful for the treatment of tumors that regulate it.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> SAMSUNG LIFE WELFARE FOUNDATION <120> COMPOSITIONS, DNA CHIPS FOR DIAGNOSIS OF CANCER CONFIRMING LIVIN          OVER-EXPRESSION AND PHARMACEUTICAL COMPOSITION FOR TREATING          CANCER <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1322 <212> DNA <213> Homo sapiens <400> 1 ccctgggata ctcccctccc agggtgtctg gtggcaggcc tgtgcctatc cctgctgtcc 60 ccagggtggg ccccgggggt caggagctcc agaagggcca gctgggcata ttctgagatt 120 ggccatcagc ccccatttct gctgcaaacc tggtcagagc cagtgttccc tccatgggac 180 ctaaagacag tgccaagtgc ctgcaccgtg gaccacagcc gagccactgg gcagccggtg 240 atggtcccac gcaggagcgc tgtggacccc gctctctggg cagccctgtc ctaggcctgg 300 acacctgcag agcctgggac cacgtggatg ggcagatcct gggccagctg cggcccctga 360 cagaggagga agaggaggag ggcgccgggg ccaccttgtc cagggggcct gccttccccg 420 gcatgggctc tgaggagttg cgtctggcct ccttctatga ctggccgctg actgctgagg 480 tgccacccga gctgctggct gctgccggct tcttccacac aggccatcag gacaaggtga 540 ggtgcttctt ctgctatggg ggcctgcaga gctggaagcg cggggacgac ccctggacgg 600 agcatgccaa gtggttcccc agctgtcagt tcctgctccg gtcaaaagga agagactttg 660 tccacagtgt gcaggagact cactcccagc tgctgggctc ctgggacccg tgggaagaac 720 cggaagacgc agcccctgtg gccccctccg tccctgcctc tgggtaccct gagctgccca 780 cacccaggag agaggtccag tctgaaagtg cccaggagcc aggaggggtc agtccagccg 840 aggcccagag ggcgtggtgg gttcttgagc ccccaggagc cagggatgtg gaggcgcagc 900 tgcggcggct gcaggaggag aggacgtgca aggtgtgcct ggaccgcgcc gtgtccatcg 960 tctttgtgcc gtgcggccac ctggtctgtg ctgagtgtgc ccccggcctg cagctgtgcc 1020 ccatctgcag agcccccgtc cgcagccgcg tgcgcacctt cctgtcctag gccaggtgcc 1080 atggccggcc aggtgggctg cagagtgggc tccctgcccc tctctgcctg ttctggactg 1140 tgttctgggc ctgctgagga tggcagagct ggtgtccatc cagcactgac cagccctgat 1200 tccccgacca ccgcccaggg tggagaagga ggcccttgct tggcgtgggg gatggcttaa 1260 ctgtacctgt ttggatgctt ctgaatagaa ataaagtggg ttttccctgg aggtacccag 1320 ca 1322  

Claims (15)

A composition for diagnosing a tumor containing a livin gene or a fragment having a continuous nucleotide sequence of 25 to 100 bp derived from the gene, and confirming whether overexpression of livin. The diagnostic composition of claim 1, wherein the tumor is pediatric leukemia. The diagnostic composition according to claim 2, wherein the pediatric leukemia is pediatric acute lymphoblastic leukemia. The diagnostic composition according to claim 1, wherein the diagnosis is a diagnosis for a prognosis after tumor treatment. A diagnostic composition for a tumor containing an antibody to a ribin protein, wherein the tumor is overexpressed. The diagnostic composition of claim 5, wherein the tumor is pediatric leukemia. The diagnostic composition according to claim 6, wherein the pediatric leukemia is pediatric acute lymphocytic leukemia. The diagnostic composition according to claim 5, wherein the diagnosis is a diagnosis for a prognosis after tumor treatment. A diagnostic DNA chip for a tumor containing a ribin gene or a fragment having a nucleotide sequence of 25 to 100 bp derived from the gene, wherein the tumor is overexpressed. 10. The diagnostic DNA chip of claim 9, wherein the tumor is pediatric leukemia. The diagnostic DNA chip of claim 10, wherein the pediatric leukemia is pediatric acute lymphocytic leukemia. 10. The diagnostic DNA chip of claim 9, wherein the diagnosis is a diagnosis of a prognosis after tumor treatment. A pharmaceutical composition for the treatment of tumors, which contains ribin as an active ingredient and controls overexpression of ribine. The pharmaceutical composition of claim 13, wherein the tumor is pediatric leukemia. The pharmaceutical composition of claim 14, wherein the pediatric leukemia is pediatric acute lymphoblastic leukemia.

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