KR20060057992A - ｐ38-DX2 and uses thereof - Google Patents

Abstract

Provided herein is a variant p38-DX2 deficient in exon 2 of p38 specifically expressed in lung cancer or liver cancer tissue, which is used as a diagnostic marker for lung cancer or liver cancer to diagnose lung cancer or liver cancer, and inhibits p38-DX2 to lung cancer Or treating or preventing liver cancer.

p38, p38-DX2, lung cancer, liver cancer, diagnostic marker

Description

ｐ38-DX2 and its use {p38-DX2 and its use}

Figure 1 shows the functional significance of p38 and the interaction of p38 with Smad2 / 3 in TGF-β signaling.

2 shows the mechanism of action of p38 in TGF-β signaling.

Figure 3 shows the difference in expression of p38 and the generation of exon2-deleted form of p38.

4 shows the effect of p38-DX2 on the intracellular stability of p38.

FIG. 5 shows the disruptive effects of p38-DX2 on TGF-β signaling and cell growth regulation.

6 shows the relationship between lung cancer formation and p38.

Figure 7 shows the results of measuring the Smad2 domain related to the interaction with p38.

8 shows the exon sequence of the human p38 gene and the primer position in p38 cDNA.

Figure 9 shows the results of looking down the expression of p38 and the production of p38-DX2 in cancer cell lines.

10 We examined p38-DX2 production and inhibition of p38 in lung cancer tissues.

11 shows that p38-DX2 is expressed in liver cancer.

12 shows the p38-DX2 expression vector.

The present invention relates to variant p38-DX2, which is deficient in exon 2 of p38 and its use. In particular, the present invention relates to p38-DX2, its use as a diagnostic marker and anticancer use using inhibitors thereof.

Lung cancer or liver cancer is a cancer with a high incidence and a high mortality rate worldwide. Cancer can be diagnosed through X-rays, computed tomography, bronchoscopy, radiologic examinations, and bronchoscopy. However, these tests can determine the extent of anatomical progression, but cannot accurately assess cell physiological or molecular genetic status.

Accordingly, various attempts have been made to diagnose lung cancer or liver cancer using markers.

Korean Patent Application No. 10-1998-0038212 relates to a method for confirming metastasis of lung cancer, which measures the expression of mitogen activated protein kinase phosphatase-1 (MKP-1) in lung cancer tissues. It describes a method for diagnosing local metastatic lung cancer. International patent WO04005891 describes a method for diagnosing lung cancer using genes such as OE372, ATP5D, B4GALT, Ppase, GRP58, GSTM4, P4HB, TPI, and UCHLI as lung cancer diagnostic markers. US patent US6117981 describes a method for the diagnosis and treatment of non-small cell lung cancer and ovarian cancer using monoclonal antibodies targeting LCGA. European patent EP0804451 describes a method for diagnosing and treating lung cancer using lung cancer specific antigen HCAVIII. In addition, US Pat. No. 6,759,508, US Pat. No. 6,746,846, US Pat. No. 6,737,514, European Patent EP0621480 and the like describe lung cancer diagnostic markers.

Korean Patent Application No. 10-2000-0040609 is a sandwich assay using lectin as a capture protein and / or a probe protein to measure the concentration of acai allosaccharide protein to determine liver cirrhosis and liver cancer. It describes a method for early diagnosis of such liver disease. Korean Patent Application No. 10-2002-0035260 discloses long chain fatty acid-coenzyme A ligase 4, panesyl diphosphate synthase, syndecane 2, imopamil-binding protein, antigenically expressed antigens in melanoma, and histidine ammonia- It describes that it can be usefully used for diagnosing human liver cancer by using a composition for diagnosing human liver cancer including lyase. European Patent EP0334962 describes a method for diagnosing liver cancer by comparing UDP-N-acetylglucusamine and glycoprotein N-acetylglucosamine transferase content. EP0339097 also describes a method for diagnosing liver cancer by measuring the amount of collagenase inhibitor present in serum, plasma, and synovial fluid by a sandwich assay method.

However, the lung cancer or liver cancer markers that have been discovered so far are low in accuracy because they use a gene expressed in normal cells as a marker to determine whether cancer occurs by comparing the difference in expression levels of marker genes in normal cells and cancer cells. In view of its usefulness as a specific lung cancer or liver cancer marker.

Under this background, the present inventors can identify whether p38-DX2, which is a deletion variant of exon 2, is not expressed at all in normal cells but only in lung cancer or liver cancer cells, and thus highly reliable when used as a marker. In addition, since p38-DX2 expression is a direct cause of cancer, it was confirmed that the use of an antibody specific for p38-DX2 protein, a siRNA or antisense nucleic acid specific for p38-DX2 mRNA can effectively treat cancer. Was completed.

It is an object of the present invention to provide a p38-DX2 protein in which the region of exon 2 of the p38 protein is deleted.

Another object of the present invention is to provide a nucleic acid molecule encoding p38-DX2 protein.

Another object of the present invention is to provide a recombinant vector comprising a nucleic acid molecule encoding a p38-DX2 protein.

Still another object of the present invention is to provide a transformant transformed with the recombinant vector.

Still another object of the present invention is to provide a method of preparing p38-DX2 protein by culturing the transformant.

Another object of the present invention is to provide an antibody specific for the p38-DX2 protein.

It is still another object of the present invention to provide a lung or liver cancer diagnostic kit comprising an antibody against p38-DX2 protein.

It is still another object of the present invention to form an antigen-antibody complex by contacting an antibody specific for the p38-DX2 protein with a detection sample, and then comparing the amount of the antigen-antibody complex with a control, to diagnose a lung or liver cancer marker p38-DX2. It is to provide a method for detecting a protein.

Another object of the present invention is to provide siRNA nucleic acid molecules specific for mRNA of p38-DX2 protein.

Still another object of the present invention is to provide a pharmaceutical composition for treating lung cancer or liver cancer comprising siRNA nucleic acid molecules specific for mRNA of p38-DX2 protein.

It is another object of the present invention to provide a method for treating lung cancer or liver cancer by administering siRNA nucleic acid molecules specific for mRNA of p38-DX2 protein.

Another object of the present invention is to provide an antisense nucleic acid having a sequence complementary to the mRNA of the protein of p38-DX2.

Another object of the present invention to provide a pharmaceutical composition for treating lung cancer or liver cancer comprising an antisense nucleic acid having a sequence complementary to the mRNA of the protein of p38-DX2.

Another object of the present invention is to provide a method for treating lung cancer or liver cancer by administering an antisense nucleic acid having a sequence complementary to the mRNA of the protein of p38-DX2.

Still another object of the present invention is to provide a method for detecting mRNA of p38-DX2, a diagnostic marker for lung cancer or liver cancer, using a primer or probe that distinguishes mRNA of p38 protein from mRNA of p38-DX2 protein.

Another object of the present invention is to provide a method for screening inhibitors that inhibit heterodimer formation of p38-DX2 protein and p38 protein.

Another object of the present invention is to cultivate cells expressing p38-DX2 and to treat the candidate inhibitors with the cells, and then compare the control and mRNA or protein levels to screen inhibitors that inhibit p38-DX2 protein expression. To provide.

Detailed description of the drawings

Figure 1 shows the functional significance of p38 and the interaction of p38 with Smad2 / 3 in TGF-β signaling. The effects of TGF-β on cell proliferation (a), colony formation (b), cell cycle progression (c), and nuclear migration (d) of Smad2 and Smad3 in p38 ^{+ / +} and p38 ^{− / −} MEFs were measured. a. Cells were incubated for 6 hours at TGF-β concentrations of 2, 4 and 4 ng / ml. When thymidine incorporation is set to 1 in TGF-β untreated cells, the numerical value represents the average of four independent experimental values. b. p38 ^{+ / +} and p38 ^{− / −} MEFs (14.5d) were incubated for 4 hours in the presence of TGF-β (2 ng / ml), fixed with formaldehyde, and colonized by Giemsa staining. c. After 24 hours of treatment with MEFs with TGF-β, the extent of the G0 / G1 phase cells was examined by flow cytometry. d. MEFs were treated with TGF-β (2ng / ml) for 1 hour. Smad2 and Smad3 were reacted with their specific antibodies and then examined with FITC-conjugated antibodies (green). Nuclei were stained with PI (red). e. The interaction of p38 with Smad2 and Smad3 was examined by co-immunoprecipitation with antibodies against Smad2 and Smad3.

2 shows the mechanism of action of p38 in TGF-β signaling. a. After treatment with A549 cells with TGF-β (2 ng / ml), the harvested and extracted proteins were immunoprecipitated with anti-Smad 2 or Smad3 antibodies and coprecipitated p38 was measured with anti-p38 antibodies. WCL represents protein western blot of whole cell lysate. b. Expression of the target genes of TGF-β, p15, p21, and PAI-1 was examined by RT-PCR in control and p38-transformed DU145. c. p38 ^-/- shows the effect of p38 on the phosphorylation of Smad2 in MEFs. d. Individual domains of Smad2 (Mad-homologous domains, MH1, MH2 and linker) were expressed with LexA fusion proteins and interaction with B42-fused p38 was observed by blue colony formation in X-gal-containing yeast medium. e. TGF-β-dependent interaction of Smad2 with TGF-β receptor was measured by co-immunoprecipitation. MEFs were treated with TGF-β at 37 ° C and incubated at 8 ° C before immunoprecipitation. The binding of TGF-β receptor with Smad2 was observed by immunoblotting with anti-TβRI antibody. f. Hourly trends in total Smad2, phosphorylated Smad2 (p-Smad2), and p38 levels after TGF-β treatment in p38 ^{+ / +} and p38 ^{− / −} MEFs.

Figure 3 shows the difference between the expression of p38 and the generation of exon2-deleted form of p38. P38 levels in various cancer cell lines were compared by Western blot (a), flow cytometry (b). A549, NCI-H460, -H322, and H290 are lung cancer cell lines, and DU145 and HCT116 are prostate and colorectal cancer cell lines, respectively. b. "Negative" refers to DU145 treated with secondary antibodies only. 2000 cells of each cell line were analyzed. c. Transcription levels of p38 were compared by RT-PCR using different primer pairs. Transcripts through exons 3-4 and 1-3 were generated using primers 6/7 and 1/5 (FIG. 8B), respectively. GAPDH was used as loading control. Small primer p38 transcripts were generated using primer pairs corresponding to transcripts of exons 1-3. This small sized transcript is a different splicing form of p38 (p38-DX2) lacking exon 2. These transcripts are produced only in cell lines showing low-p38 levels when RT-PCR between exons 1 and 3 using primers DX2-F specific to the conjugation sequence and another primer (DX2-B) (FIG. 8B) It became. d. TGF-β-dependent induction and nuclear migration of p38 was investigated by immunofluorescence staining after incubation for 2 hours with TGF-β. e. The effect of TGF-β on the proliferation of the indicated cells was compared by [ ³ H] thymidine insertion ( n = 4). f. TGF-β-dependent induction of the gene of interest, p21 and PAI-1 was compared by RT-PCR after 2 hours treatment with TGF-β.

4 shows the effect of p38-DX2 on the intracellular stability of p38. a. After transforming DX2 into DU145 cells treated or not treated with TGF-β for 2 hours, p38 levels were measured by Western blot. Expression of c-myc, DX2 and GAPDH (control) was examined by RT-PCR. b. After transforming DX2 and the empty vector with DU145, the effect on TGF-β-dependent cell growth inhibition was examined by thymidine insertion. (N = 4) c. The interaction between p38-F and -DX2 was measured by the East to Hybrid Assay (Rho, SB et al., Proc Natl Acad Sci USA 96, 4488-93, 1999). d. p38-F and -DX2 were synthesized in-vitro translation in the presence of [35S] methionine, mixed with GST-p38-F or -CDK2 (control) and precipitated with glutathione-sepharose. The precipitated protein was separated by SDS-PAGE and detected by autoradiography. e. Interaction with p38-F or -DX2 with FUSE-binding protein (FBP) (Kim, MJ et M., Nat. Genet. 34, 330-336, 2003) and Smad2 measured by East to Hybrid It was. f. The effect of the proteasome inhibitor, ALLN (50 μM, 4 hours) on the levels of p38-F and -DX2 was determined by Western blot using anti-p38 antibody in DX2-produced H322 cells. DX2 mode is that of this. The bitpart synthesized counterpart and gel phase co-migration were identified (data not available). g. P38 increase after treatment with ALLN (20 μM, 2 hours) was examined by immunofluorescence staining with anti-p38 antibody in H322 cells. h. Myc-tagged DX2 was transformed into ALLN treated DU145 cells, immunoprecipitated p38 with anti-p38 antibody and immunoblotted with ubiquitinated p38 molecule with anti-ubiquitin antibody (Ubi).

FIG. 5 shows the disruptive effects of p38-DX2 on TGF-β signaling and cell growth regulation. a. After transforming DX2 (or empty vector) with MEFs, the effect on cell growth was examined. Cells and clones were examined by light microscopy (top) and Giemsa staining (bottom), respectively. b. Nucleotide sequences encoding three kinds of siRNAs specific for DX2 are shown. c. Three kinds of siRNA expressing vectors were prepared and transformed into H322 and H460 cell lines, which are cancer cell lines, and their effects on the phosphorylation of normal p38 and smad2 were investigated. d. When siRNAs 4 and 5 were transformed into H322 cells, the degree of cell death by TGF-β (left) and the degree of cell cycle arrest (right) were examined. e. Vectors expressing siRNA 4 (si-DX2) targeting p38-DX2 were introduced into H322 and the effect on DX2 transcription inhibition was measured by RT-PCR (Phase). si-DX2 did not affect full-length p38 transcription, as can be seen in the RT-PCR of the transcript corresponding to exon 3-4. The effect of si-DX2 on phosphorylation and p38 expression of Smad2 was measured by Western blot (bottom). The effect of si-DX2 on the repair of TGF-β signaling was measured by immunofluorescence (f) of Smad2, TGF-β dependent reporter assay under 3TP promoter (g) and growth arrest (h) using H322. . f. p-Smad2 and nuclei were stained 30 minutes with FITC-conjugated secondary antibody (green) and PI (red) 30 minutes after TGF-β treatment. The transfection of si-DX2 increases p-Smad2 and places it in the nucleus.

6 shows the relationship between lung cancer formation and p38. a. Lung tumor formation was observed at time intervals after intraperitoneal administration of benzo- (α) -pyrene with p38 ^{+ / +} and p38 ^+/− mice. “N” represents the number of slaughtered mice b.Histochemical characteristics and p38 levels in lung tumors isolated from p38 ^+/− mice were immunofluorescent (IF) stained with H & E (left) and anti-p38 antibodies ( Right) “T” and “N” mean tumor and normal site, respectively. Nuclei and p38 appear in red (PI staining) and green (FITC). c. Immunofluorescence staining of p38 at normal and tumor sites isolated from lung cancer patients. Normal tissues were isolated from non-tumoral inflammatory tissues. d. Total RAN was isolated from the tissue used in (c) and RT-PCR was performed with DX-2 specific primers. Normal and tumor tissues of the same patient (expressed by code number) were subjected to immunofluorescence staining (e) and RT-PCR (f) using anti-p38 antibodies. f. The exon 4 region of p38 and GAPDH were used as controls.

Figure 7 shows the results of measuring the Smad2 domain related to the interaction with p38. The cDNA encoding p38 or CDK2 was combined with the EcoRI and XhoI sites of pGEX4T-1 and expressed in E. coli BL21 (DE3) as a GST-fusion protein, and then the fusion protein was isolated according to the manufacturer's instructions. Different Smad2 deletion fragments were synthesized by in vitro translation in the presence of [35S] methionine using the TNT coupled transcription kit (Promega). GST fusion proteins bound to glutathione-sepharose beads were prepared in binding buffer of PBS buffer (pH 7.4) containing 0.5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 1% Triton X-100 [ 35S] methionine-labeled Smad fragments. The binding mixture was incubated overnight with rotation at 4 ° C. and then washed four times with binding buffer containing 0.5% Triton X-100. After addition of SDS sample buffer, bound proteins were boiled and eluted to separate by SDS gel electrophoresis. The presence of the Smad2 fragment was confirmed by autoradiography.

8 shows the exon sequence of the human p38 gene and the primer position in p38 cDNA. a. The p38 gene consists of four exons (marked in blue) that encode polypeptides of the indicated size. b. Schematic showing the position and sequence of the primers used to generate cDNA penetrating different regions of p38.

Figure 9 shows the results of looking down the expression of p38 and the production of p38-DX2 in cancer cell lines. a. To compare p38 levels between DX2-positive and -negative cells, DU145 and H460 cells were cultured in one dish and subjected to immunofluorescence with anti-p38 antibody (green). Because DU145 cells express high levels of p53 due to mutations, H460 cells have low levels because they have a natural type of p53, so the two cell lines were divided by p53 immunofluorescence staining (red). Cells were treated with TGF-β for 2 hours and fixed with methanol. b. To examine the effect of p38-DX2 on p38 expression, p38 levels were examined by flow cytometry. 2 μg / ml of the empty vector or DX2 were transformed into DU145 cells and incubated for 24 hours. Cells were fixed with 70% ethanol and then reacted with anti-p38 antibody followed by FITC-conjugated secondary antibody. c. The effect of p38-DX2 on TGF-β-dependent cell cycle arrest was compared by flow cytometry. The ratio of cells on G0 / G1 in the DU145 cells transformed with the empty vector increased, whereas not in DX2-transformed cells. Black and blue lines represent cells untreated and treated with TGF-β, respectively. d. P38 levels were compared in H460 cells treated with 2 hours untreated (control) or treated with 10 μM ALLN. "Negative" refers to cells incubated with FITC-conjugated secondary antibodies only.

10 We examined p38-DX2 production and inhibition of p38 in lung cancer tissues. a. After lungs were isolated from p38 ^{+ / +} and p38 ^+/− mice injected with benzopyrene, RNA was isolated from each lung to determine production of p38-DX2 by RT-PCR. All lungs producing DX2 had tumors in the lungs (marked with +). b. Normal and tumor tissues were isolated from each of the 4 patients and p38 levels were compared by immunofluorescence staining (green) with anti-p38 antibodies. Nuclei were observed with PI staining (red). Histological properties of the isolated tissues were observed by H & E staining. Tissue sites indicated by the arrows were immunostained as seen from the top.

11 shows that p38-DX2 is expressed in liver cancer. a. Liver cancer and p38-DX2 formation in human tissues were examined by RT-PCR. b. The reduction of p38 levels in liver cancer tissues was examined by tissue staining.

12 shows the p38-DX2 expression vector.

In relation to the p38 protein, we found that in the previous study, genetic breakdown of p38 induced over-expression of c-myc, resulting in overgrowth of alveolar epithelial cells in the lung, resulting in neonatal lethality. Molecular and cytological analysis has revealed that p38 is induced by TGF-β and migrates to the nucleus to inhibit the expression of c-myc (MJ Kim, B.-J. Park, Y.). -S. Kang, HJ Kim, J.-H. Park, JW Kang, SW Lee, JM Han, H.-W. Lee, S. Kim, Nat. Genet. 34, 330-336, 2003).

Following the above studies, we found that p38 is a novel cancer suppressor that functions to enhance TGF-β signaling through direct interaction with Smad2 / 3. In addition, it was confirmed that p38-DX2, which is a variant of p38 lacking exon 2, was specifically expressed in cancer cell lines and tissues: RT-PCR was performed for the presence of p38-DX2, which is a p38 variant lacking exon 2 It was confirmed that the band appears differently depending on the p38-specific primer combination. When primer pairs derived from p38 cDNA containing exons 3 and 4 were used for RT-PCR, no decrease in p38 transcription was observed in cells with low p38 levels in the western blot, but transcripts containing exons 1 to 3 When RT-PCR was performed with the primers derived from, small transcripts appeared with the expected transcripts. Sequencing the transcripts appearing as small bands revealed that all of the 69 amino acids corresponding to exon 2 of p38 were missing variants of p38. RT-PCR using primers specific for the conjugation sequences of exon 1 and exon 3 confirmed that PCR products were produced only in cell lines that exhibit low p38 levels and produce smaller transcripts in addition to standard p38 transcripts. Once again demonstrating the presence of the p38 variant.

In addition, we found that p38-DX2-transformed cells dramatically reduced p38 levels irrespective of TGF-β, resulting in the loss of p38-DX2 production resulting in loss of p38 activity. Transformation resulted in aberrant-functions of TGF-β signaling such as increasing c-myc expression, preventing nuclear migration of p38 and stopping cell growth. p38-DX2 forms a heterodimer with p38, in which case p38 decomposes rapidly as ubiquitination, leading to a decrease in p38 levels, which is closely linked to cancer formation and progression Proved. The production of p38-DX2 has been observed in liver cancer as well as lung cancer.

In one embodiment, the present invention provides a p38-DX2 protein, which is a p38 variant lacking exon 2 specifically expressed in lung cancer or liver cancer.

The p38-DX2 protein of the present invention is a variant in which the region of exon 2 is deleted in the p38 protein sequence, and the sequence of p38 protein (312aa version: AAC50391.1 or GI: 1215669; 320aa version: AAH13630.1, GI: 15489023, BC013630). .1) (312aa version: Nicolaides, NC, Kinzler, KW and Vogelstein, B. Analysis of the 5 'region of PMS2 reveals heterogeneous transcripts and a novel overlapping gene, Genomics 29 (2), 329-334 (1995) // 320 aa version: Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences, Proc. Natl. Acad. Sci. USA 99 (26), 16899-16903 (2002)). The region corresponding to exon 2 is deleted. Korean Patent Application No. 10-2003-0018424, filed by the inventors, describes the cancer therapeutic effect of p38 protein, the description of which is described herein in this patent document.

The p38-DX2 protein described above includes a protein in which a region of exon 2 is deleted in p38 of the entire sequence, and is substantially equivalent to p38 in a p38 equivalent (a modification by substitution, deletion, insertion, or a combination of amino acid sequences). Functional equivalents having, or functional derivatives having modifications that increase or decrease physicochemical properties, but which have a substantially equivalent activity to p38).

In the present invention, “deleting the region of exon 2” in the p38 protein sequence means that the variant resulting from the partial or total loss of the amino acid sequence (amino acids 46-114) of the exon 2 region in the p38 protein forms a heterodimer with the p38 protein. To interfere with the normal functioning of p38 and to promote degradation. Thus, the p38-DX2 protein may have all of the amino acid sequences of exon 2 of the p38 protein deleted, or some of these regions in exon 1, exon 3, exon 4 or both of these regions, including the amino acid sequence of this region, or exon 2 Only part of the amino acid sequence of contains a deleted protein. Preferably, the p38-DX2 protein of the invention is a protein in which all of the amino acid sequence of exon 2 of the p38 protein is deleted. More preferably, the p38-DX2 protein of the present invention is a protein having the amino acid sequence of SEQ ID NO.

In addition, the p38-DX2 protein of the present invention, as well as proteins having its native amino acid sequence, as well as its amino acid sequence variants are also included within the scope of the present invention. A variant of the p38-DX2 protein refers to a protein in which the p38-DX2 native amino acid sequence and one or more amino acid residues have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like.

The p38-DX2 protein or variant thereof may be extracted from nature or synthesized (Merrifleld, J. Amer. Chem. Soc .. 85: 2149-2156, 1963) or prepared by recombinant methods based on DNA sequences ( Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989). When using a recombinant technique, a nucleic acid encoding p38-DX2 is inserted into an appropriate expression vector, the host cell is cultured to express p38-DX2 in a transformant transformed with the recombinant expression vector, and then It can be obtained by recovering p38-DX2.

As described above, the present inventors have found that p38-DX2 protein is specifically expressed in lung cancer or liver cancer tissue, and the detection of such protein can be used to diagnose lung cancer or liver cancer, so that p38-DX2 is a marker of lung cancer or liver cancer. It can be used as a diagnostic marker.

In another aspect, the invention provides nucleic acid molecules encoding the p38-DX2 protein.

A nucleic acid sequence encoding a p38-DX2 protein as described above may be mutated by substitution, deletion, insertion or combination thereof, as long as it encodes a protein having equivalent activity. The p38-DX2 protein of SEQ ID NO: 2 above is preferably encoded by a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1. The sequence of such nucleic acid molecules may be single or double stranded, and may be DNA molecules or RNA (mRNA) molecules.

Nucleic acid sequences encoding the p38-DX2 protein of the present invention can be isolated from nature or produced artificially through synthetic or genetic recombination methods. The p38-DX2 protein encoding a nucleic acid sequence of the present invention can be operably linked to a vector capable of expressing it to provide a p38-DX2 protein.

In another aspect, the invention provides a recombinant vector comprising a nucleic acid molecule encoding a p38-DX2 protein.

As used herein, a “recombinant vector” refers to a gene construct that is capable of expressing a target protein or target RNA in a suitable host cell and includes essential regulatory elements operably linked to express the gene insert.

As used herein, the term “operably linked” refers to the functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the encoding nucleic acid sequence.Operative linkage with recombinant vectors may be accomplished using genetic recombination techniques well known in the art. Site-specific DNA cleavage and ligation using enzymes generally known in the art.

Vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The expression vector also includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin.

The signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia spp., And an α-amylase signal sequence and a subtilisin signal sequence when the host is Bacillus. The signal sequence, the SUC2 signal sequence, and the like, when the host is an animal cell, may use an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, and the like, but are not limited thereto.

In another aspect, the present invention provides a transformant transformed with the recombinant vector.

Transformation includes any method of introducing nucleic acids into an organism, cell, tissue or organ, and can be carried out by selecting appropriate standard techniques according to the host cell as known in the art. These methods include electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation with silicon carbide fibers, agro bacterial mediated transformation, PEG, dextran sulfate, lipofect Such as but not limited to.

Depending on the host cell, the expression level and the expression of the protein are different, so you can select and use the most suitable host cell for the purpose. Host cells include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. Prokaryotic host cells such as, but are not limited to. In addition, fungi (e.g. Aspergillus), yeast (e.g. Pichia pastoris, Saccharomyces cerevisiae, Schizocaromaces ( Cells derived from higher eukaryotes, including lower eukaryotic cells such as Schizosaccharomyces and Neurospora crassa, insect cells, plant cells, mammals and the like, can be used as host cells.

In another aspect, the present invention provides a method for producing p38-DX2 protein by culturing the transformant.

Cultivation of the transformants is carried out under appropriate conditions that enable the expression of the desired protein p38-DX2, which can be carried out according to methods well known to those skilled in the art.

Proteins expressed in the transformants can be purified in a conventional manner, for example, salting out (eg ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (protein fraction precipitation with acetone, ethanol, etc.), dialysis Techniques such as column chromatography such as gel filtration, ion exchange, reverse phase column chromatography, and ultrafiltration may be applied alone or in combination to purify the p38-DX2 protein of the invention (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990)).

In the present invention, the term "lung cancer or liver cancer diagnostic marker" is expressed in tissues and cells related to lung cancer or liver cancer and a substance capable of confirming the onset of lung cancer or liver cancer, preferably normal tissue and lung cancer or liver cancer tissue by confirming the expression thereof. Refers to an organic biomolecule such as a protein or mRNA showing a significant difference. For the purposes of the present invention, p38-DX2, which is not expressed in normal cells but specifically expressed in lung cancer or liver cancer tissues and cells, is used as a diagnostic marker for lung cancer or liver cancer. Preferably, lung cancer or liver cancer can be diagnosed by checking the expression of p38-DX2 at the mRNA level and / or protein level.

In another aspect, the present invention provides antibodies specific for p38-DX2.

As used herein, the term “antibody” refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically recognizes p38-DX2, distinct from p38, and includes both polyclonal and monoclonal antibodies.

Since the p38-DX2 protein has been identified as described above, generating antibodies using the same can be easily prepared using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for injecting the p38-DX2 protein antigen described above into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.

Monoclonal antibodies are known in the art for fusion methods (see Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519), recombinant DNA methods (US Pat. No. 4,816,567) or phage antibody libraries (Clackson et al, Nature, 352: 624-628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991).

Antibodies used in the detection of p38-DX2 proteins of the invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function and includes Fab, F (ab '), F (ab') 2 and Fv.

The antibody specific for p38-DX2 of the present invention can be used not only for the diagnosis of lung cancer or liver cancer, but also for the treatment of lung cancer or liver cancer by inhibiting the activity of p38-DX2 by administration to a patient. When used as a therapeutic antibody, the antibody may be coupled (eg covalently) with an existing therapeutic agent directly or indirectly through a linker or the like.

 Therapeutic agents that can be combined with antibodies include, but are not limited to, radionuclides, drugs, lymphokines, toxins, heterozygous antibodies, and the like. Radionuclides include 131I, 90Y, 105Rh, 47Sc, 67Cu, 212Bi, 211At, 67Ga, 125I, 186Re, 188Re, 177Lu, 153Sm, 123I, 111In and the like. Drugs include methotrexate and adriamycin, and lymphokines include interferon. Toxins include lysine, abrin, and diphtheria. And heterofunctional antibodies, which are antibodies that bind to other antibodies and bind their complexes to both cancer cells and agonist cells (eg, K cells such as T cells).

The antibody can be administered by itself or in a composition comprising the antibody.

For therapeutic compositions, they are prepared in appropriate formulations, including acceptable carriers depending on the mode of administration. Formulations suitable for the mode of administration are known and typically comprise a surfactant that facilitates movement through the membrane. Such surfactants are steroid derived or cationic lipids such as N- [1- (2,3-dioleoyl) propyl-N, N, N-trimethylammonium chloride (DOTMA), or cholesterol hemisuccinate And various compounds such as phosphatidyl glycerol.

Compositions comprising the antibodies of the invention can be administered in pharmaceutically effective amounts to treat cancer cells or their metastases. The pharmaceutical composition may be administered single or multiple. The composition comprising the antibody is administered by a suitable method including parenteral, subcutaneous, intraperitoneal, pulmonary, and intranasal, and if necessary for local immunosuppressive treatment, including intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like. The pH of the formulation is balanced to antibody stability (chemical and physical stability) and is appropriate for the patient to be administered, generally in the range of pH 4 and pH 8. In addition, appropriate formulations can be devised by appropriately modifying other techniques for administration such as transdermal administration and oral administration. Typical dosage levels can be optimized using standard clinical techniques.

In addition, the antibody of the present invention can be administered in the form of a nucleic acid encoding the antibody so that the antibody is produced in the cell (WO96 / 07321).

In another aspect, the present invention provides a lung or liver cancer diagnostic kit comprising an antibody specific for p38-DX2.

Lung cancer or liver cancer diagnostic kits of the present invention include not only antibodies that selectively recognize p38-DX2 protein, but also tools, reagents, and the like commonly used in the art for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labeling materials capable of producing detectable signals, solubilizers, detergents, buffers, stabilizers, and the like. If the labeling substance is an enzyme, it may include a substrate and a reaction terminator capable of measuring enzyme activity. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, Polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.

Lung or liver cancer diagnostic kits of the invention may take the form of, but not limited to, ELISA plates, dip-stick devices, immunochromatography test strips and radiation split immunoassay devices, and flow-through devices. Can be.

In another aspect, the present invention provides a method for detecting a lung cancer or liver cancer diagnostic marker protein by contacting an antibody specific for p38-DX2 with a detection sample, forming an antigen-antibody complex and comparing the amount of antigen-antibody complex formation with a control. To provide.

As used herein, the term “detection sample” refers to a biological sample such as tissue, cells, whole blood, serum, plasma, saliva, semen, cerebrospinal fluid, or urine, in which a difference in the expression level of a marker protein can be detected by lung cancer or liver cancer. And prepared by treatment in a manner well known in the art.

As used herein, the term “antigen-antibody complex” refers to a combination of a p38-DX2 protein in a biological sample and an antibody that specifically recognizes it. Experimental methods to examine antigen-antibody complex formation include tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assay, immunodiffusion assay (Immunodiffusion). assay, Complement Fixation Assay, FACS, protein chip, and the like, but are not limited thereto.

As used herein, the term “lung cancer or liver cancer diagnostic marker protein detection” refers to the presence or absence of p38-DX2 protein, a diagnostic marker for lung cancer or liver cancer, by qualitatively or quantitatively measuring the signal size of a detection label bound to an antigen-antibody conjugate. Means checking the amount.

Labels that enable qualitative or quantitative measurement of antigen-antibody complex formation include, but are not limited to, enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes. . Enzymes available as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, and glucose oxy Multidase, Hexokinase and GDPase, RNase, Glucose Oxidase and Luciferase, Phosphofructokinase, Phosphoenolpyruvate Carboxylase, Aspartate Aminotransferase, Phosphopyruvate Decarboxylase, β- Latamases and the like, but are not limited thereto. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN) ⁸ , [Os (bpy) ₃ ] ²⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like.

Lung cancer or liver cancer can be diagnosed by examining whether there is a significant difference between the amount of antigen-antibody complex formation in the control and the detection sample as an absolute (e.g. μg / ml) or relative (e.g. relative intensity of signal) difference. .

In another aspect, the invention provides a small interfering RNA (siRNA) nucleic acid molecule specific for mRNA of p38-DX2.

In the present invention, the term “siRNA” refers to a short double-chain RNA capable of inducing RNA interference (RNAi) through cleavage of a specific mRNA, and a sense RNA strand having a sequence homologous to mRNA of a target gene. It is composed of antisense RNA strands having complementary sequences, and siRNA is provided as an efficient gene knockdown method or gene therapy method because it can suppress expression of a target gene.

siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases. The siRNA terminal structure can be either blunt or cohesive, as long as the expression of the target gene can be suppressed by the RNAi effect. The cohesive end structure is possible in both a three-terminal protruding structure and a five-terminal protruding structure. The number of protruding bases is not limited. For example, the number of bases may be 1 to 8 bases, preferably 2 to 6 bases. The full length of the siRNA herein is expressed as the sum of the length of the central double chain portion and the length constituting the single chain protrusion of both ends. In addition, siRNA is a low-molecular RNA (e.g., a natural RNA molecule such as tRNA, rRNA, viral RNA or artificial RNA) in the protruding portion of one end to the extent that can maintain the expression inhibitory effect of the target gene Molecules). The siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a stem loop type structure in which one terminal portion of the double chain RNA is connected by a linker RNA. The length of the linker is not particularly limited as long as it does not interfere with pairing of stem portions.

In the present invention, the term "specific" or "specific" means the ability to inhibit only the gene of interest without affecting other genes in the cell, and is p38-DX2 specific in the present invention. The siRNA of the present invention has a sense RNA strand comprising a sequence homologous to an mRNA corresponding to the exon 1 and exon 3 boundaries of p38-DX2 and an antisense RNA strand comprising a sequence complementary thereto so as to specifically operate p38-DX2. Has

As used herein, the term "inhibition of gene expression" means that the level of mRNA and / or protein produced in the gene of interest is eliminated or reduced, which means that RNA interference (RNAi) occurs through cleavage of mRNA. : RNA interference.

The method of preparing siRNA is a method of directly synthesizing siRNA in vitro and then introducing it into a cell through a transfection process and siRNA expression vector or PCR-derived siRNA expression cassette prepared to express siRNA into the cell. There is a method of transformation or infection. The determination of how to prepare siRNAs and introduce them into cells or animals may depend on the purpose of the experiment and the cell biological function of the target gene product.

The siRNA of the present invention is not particularly limited in sequence and length as long as it can specifically reduce p38-DX2 mRNA. In a specific embodiment of the present invention, a vector expressing three types of p38-DX2 specific siRNA was prepared. Was found to reduce intracellular levels of p38-DX2 and restore p38 function and TGF-β signaling.

The siRNA specific for p38-DX2 of the present invention is a nucleic acid comprising a sequence corresponding to a site where exon 1 and exon 3 are linked to p38-DX2 mRNA. Preferably, siRNA No. 3 comprising an RNA sequence expressed from the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10, siRNA No. 4 comprising an RNA sequence expressed from the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12, or a sequence SiRNA # 5 comprising an RNA sequence expressed from the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 14. Among them, siRNAs including RNA sequences expressed from the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12 are most preferred. Single or two or more kinds of siRNA can be used in combination.

In another aspect, the invention provides antisense nucleic acid sequences complementary to mRNA of p38-DX2 protein.

As used herein, the term "antisense nucleic acid" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to a complementary sequence in the mRNA to inhibit translation of the mRNA into a protein. The antisense sequence of the present invention refers to a DNA or RNA sequence that is complementary to p38-DX2 mRNA and capable of binding to p38-DX2 mRNA, and includes translation of p38-DX2 mRNA, translocation, and maturation into the cytoplasm. maturation) or any other overall biological function, the antisense nucleic acid is 6 to 100 bases in length, preferably 8 to 60 bases, more preferably 10 to 40 bases.

The antisense nucleic acid can be modified at the position of one or more bases, sugars or backbones to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol., 5 (3): 343-55, 1995). . The nucleic acid backbone can be modified with phosphorothioate, phosphoroester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic intersaccharide linkages and the like. In addition, antisense nucleic acids may comprise one or more substituted sugar moieties. Antisense nucleic acids can include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-me pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this. In addition, the antisense nucleic acids of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cellular adsorption of the antisense nucleic acids. Cholesterol moieties, cholesteryl moieties, cholic acid, thioethers, thiocholesterols, fatty chains, phospholipids, polyamines, polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine, hexyl amino-carbonyl-oxy Fat-soluble moieties such as a cholesterol ester moiety, and the like. Oligonucleotides comprising fat-soluble moieties and methods of preparation are already well known in the art (US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid can increase stability to nucleases and increase the binding affinity of the antisense nucleic acid with the target mRNA.

The antisense nucleic acid specific for p38-DX2 of the present invention preferably includes a sequence complementary to the sequence corresponding to the site where exon 1 and exon 3 are linked to p38-DX2 mRNA.

In the case of antisense RNA, it may be synthesized in vitro in a conventional manner to be administered in vivo or to allow antisense RNA to be synthesized in vivo. One example of synthesizing antisense RNA in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin of the recognition site (MCS) is in the opposite direction. Such antisense RNA is desirable to ensure that there is a translation stop codon in the sequence so that it is not translated into the peptide sequence.

In still another aspect, the present invention provides a pharmaceutical composition for treating lung cancer or liver cancer comprising siRNA or antisense nucleic acid specific for mRNA of p38-DX2 protein and a method for treating lung cancer or liver cancer by administering the same.

Pharmaceutical compositions comprising at least one siRNA or p38-DX2 antisense nucleic acid may comprise additional substances that inhibit the proliferation of cancer cells of the patient, and also include agents that promote the influx of siRNA or antisense nucleic acid molecules, for example , Liposomes (US Pat. Nos. 4,897,355, 4,394,448, 4,235,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567, 4,247,411, 4,814,270) or cholesterol, cholate and deoxycholic acid. It may also be combined with one of the lipophilic carriers of a number of sterols. Antisense nucleic acids can also be conjugated to peptides that are taken up by cells. Examples of useful peptides include peptide hormones, antigens or antibodies and peptide toxins (Haralambid et al, WO 8903849; Lebleu et al., EP 0263740).

The pharmaceutical composition of the present invention may be administered together with a pharmaceutically acceptable carrier, and upon oral administration, a binder, a suspending agent, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, and the like In the case of injections, buffers, preservatives, analgesic agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elesir, suspensions, syrups, wafers, etc., and in the case of injections, may be prepared in unit dosage ampoules or multiple dosage forms.

The range of effective dosage of siRNA or antisense nucleic acid specific for mRNA of p38-DX2 protein contained in the pharmaceutical composition of the present invention will vary depending on various factors such as sex, severity, age, method of administration, target cell, expression level, etc. It can be easily determined by those skilled in the art.

The pharmaceutical compositions according to the invention are administered to humans and animals orally or parenterally, intravenously, subcutaneously, intranasally or intraperitoneally. Parenteral administration includes injection and drip methods such as subcutaneous injection, intramuscular injection and intravenous injection. Other pharmaceutical compositions of the present invention may be prepared according to techniques commonly used in the form of various formulations.

In another aspect, the present invention provides a method for detecting the mRNA of p38-DX2 protein for lung cancer or liver cancer using a primer or probe that distinguishes the mRNA of p38 protein and the mRNA of p38-DX2 protein.

In the present invention, the term “detection of mRNA of p38-DX2 protein” refers to the presence or absence of mRNA of p38-DX2, a diagnostic marker for lung cancer or liver cancer expressed in cells, using primers or probes specific for p38-DX2 mRNA. It can be detected.

As used herein, the term “primer” refers to a nucleic acid sequence having a short free 3 ′ hydroxyl group, which can form complementary templates and base pairs and serves as a starting point for template strand copying. Short nucleic acid sequence.

In the present invention, the term “probe” refers to a nucleic acid fragment such as RNA or DNA, which is short to several bases to hundreds of bases, which is capable of specific binding with mRNA, and is labeled to identify the presence of a specific mRNA. Can be. Probes can be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, and the like, and include biotin, FITC, rhodamine, DIG, etc. Or labeled with a radioisotope or the like.

Methods for detecting p38-DX2 mRNA using probes or primers include DNA sequencing, RT-PCR, and primer extension methods (Nikiforov, TT et al., Nucl Acids Res 22, 4167-4175 ( 1994), oligonucleotide extension assays (OLA) (Nickerson, DA et al., Pro Nat Acad Sci USA, 87, 8923-8927 (1990)), allele-specific PCR methods (Rust, S. et al., Nucl Acids Res, 6, 3623-3629 (1993)), RNase mismatch cleavage; Myers RM et al., Science, 230, 1242-1246 (1985)), single strand conformation lymorphism: SSCP; Orita M. et al., Pro Nat Acad Sci USA, 86, 2766-2770 (1989)), simultaneous analysis of SSCP and heteroduplex (Lee et al., Mol Cells, 5: 668-672 (1995)) , Modified gradient gel electrophoresis (DGGE; Cariello NF. Et al., Am J Hum Genet, 42, 726-734 (1988)), denaturing high performance liquid chromatography (D-HPLC, Underhill PA. et al., Genome Res, 7, 996-1005 (1997)), but is not limited to such.

Preferably, the method is a method of detecting lung cancer or liver cancer diagnostic marker mRNA by performing RT-PCR using a specific primer. RT-PCR is a method introduced by P. Seeburg (1986) to analyze RNA, and amplifies and analyzes cDNA obtained by reverse transcription of mRNA by PCR. At this time, the amplification step to use a primer prepared for the purpose of the present invention. For the purpose of the present invention, primers can be prepared with primers represented by two bands of different sizes corresponding to normal p38 mRNA and variant p38-DX2 mRNA, and primers showing only bands corresponding to mRNA of p38-DX2 protein, respectively. In order to appear as two bands of different sizes, two primer positions may be designated so that the portion corresponding to exon 2 is transferred. The kind of primer is not particularly limited, but in the present invention, primers of SEQ ID NO: 5 and SEQ ID NO: 6 were used. In order to prepare a primer so that only the band corresponding to the variant appears, one primer may be prepared to have a sequence of a point where the C terminal of exon 1 and the N terminal of exon 3 are connected. In the present invention, the primer of SEQ ID NO: 8 was prepared using the primer corresponding to the conjugation point, and used with the primer of SEQ ID NO: 7 to perform RT-PCR. RT-PCR is a convenient way to check whether the diagnostic marker p38-DX2 mRNA expression by checking the band pattern and whether lung cancer or liver cancer.

In another aspect, the present invention provides a method for screening inhibitors that inhibit heterodimer formation of p38-DX2 protein and p38.

Screening preferably, Yeast-two-hybrid, in vitro pull-down assays can be used.

East-to-hybrid assay was first developed by Field & Song (1989), which uses bite and prey proteins in the DNA binding and transcription activation domains of transcriptional regulatory proteins, respectively. It is a screening method that can confirm whether the two proteins (prey protein, bite protein) interaction by expressing in the form of a fusion protein, the expression of the gene regulated by the transcriptional regulator protein and the yeast grow in the nutrient deficient medium. In vitro pull-down assays confirm the interaction between two proteins by purifying tagged proteins (bite proteins) and by checking whether the protein binding partner (preprotein) that binds to the bite protein is pulled down. It is a screening method that can be. These screening methods can be performed to select inhibitors that inhibit heterodimer formation of p38-DX2 and p38.

In another embodiment, the present invention provides a method for expressing lung cancer or liver cancer diagnostic markers (protein, mRNA) in control cells which are cultured with p38-DX2 protein and treated with a candidate inhibitor and not treated with the candidate inhibitor. A method of screening inhibitors that inhibit p38-DX2 protein expression in comparison to is provided.

For screening, preferably, a method using a primer or a probe may be used to measure the mRNA expression level of a lung cancer or liver cancer diagnostic marker, and among them, RT-PCR is preferable. Inhibitors that reduce the expression of diagnostic markers can be selected for use in the treatment of lung or liver cancer as compared to the control.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Cell Culture, Compound and Cell Cycle Analysis

The cell lines used in this study were maintained in RPMI-1640 containing 10% FBS. Mouse embryonic fibroblasts (MEFs) were isolated from embryos 12.5 days to 14.5 days old and cultured in DMEM containing 20% FBS. To examine the effect of TGF-β on the cell cycle, cells were treated with 2 ng / ml TGF-β in a medium containing no serum or 1% FBS, and the cells were collected and FACS analyzed. Cell proliferation was also measured by [ ³ H] thymidine incorporation. Cells were incubated for 20 hours in serum-free medium treated with or without TGF-β, and then cultured for 4 hours in the presence of 1 μCi / ml of [ ³ H] thymidine. The inserted thymidine was quantified according to the literature (Kim, MJ et al., Nat. Genet. 34, 330-336 (2003)) using a liquid scintillation couner. TGF-β was purchased from the R & D system and anti-Smad 2, and -Smad4 antibodies were purchased from Santa Cruz.

Immunoprecipitation and Western Blot Analysis

Cells were treated with TGF-β for a period of time and proteins were extracted from the cells using RIPA buffer containing protease, isolated using 10-12% SDS-PAGE, followed by specific antibodies. Immunoblotting using the ECL system. For immune precipitation, cell lysates were treated with IgG and agarose-conjugated protein-A. After centrifugation, the supernatant was incubated for 2 hours with specific antibody and agarose-conjugated A. After washing twice with cold PBS and once with RIPA, the bound proteins were precipitated with specific antibodies and eluted to perform Western blot analysis.

 RT-PCR

Total RNA (total-RAN) was isolated according to the manufacturer's protocol (Qiagen). Freshly prepared tissue (3 × 3 × 3 mm) was cut into small pieces, mixed with 350ul lysine buffer, and homogenized with a homogenizer or syringe. After adding 350ul of 70% ethanol, the lysate was shaken up and down several times, loaded onto the column and centrifuged for 15 seconds at 13,000 RPM. After washing the column twice with wash buffer, RNA was eluted with 40ul of RNase-free DW. For reverse transcription, 1 μg of isolated RNA was used as template for p38-specific primers (FIG. 8B). After reverse transcription, dilute to DW 3 fold and 1 ul 30ul PCR reaction with 0.5 ul dMTP (2.5 mM each), 0.5 ul indicated primer (10 pM each), 1.5 ul DMSO and 0.1 ul Taq polymerase (5 U / ul) Was used.

Inhibition of p38DX2 by siRNA

In order to inhibit the expression of p38DX2, siRNAs for p38DX2 were constructed. A sequence encoding the siRNA for p38DX2 was constructed and linked to SalI and XbaI sites of the IMG-700 (Imgenex) vector and cloning confirmed by DNA sequencing. H322 cells were transformed with 2 μg of si-DX2 expressing vector. After incubation with the DNA-liposomal complex for 3 hours in 1 ml serum-free medium, 1 ml of RPMI-1640 containing 20% FBS was administered to H322 cells. An additional 20 hours of incubation followed by treatment with TGF-β for serum-free conditions or for the indicated times.

East to Hybrid Assays

CDNAs encoding human p38 and Smad2 (and deletion fragments thereof) were obtained by PCR using specific primers. PCR products of Smad2 and p38 were cleaved with EcoRI and XhoI and bound to the same sites of pEG202 (LexA) and pJG4-5 (B42), respectively (Gyuris, J et al., A human G1 and S phase protein phosphatase that associates with Cdk 2. Cell 75 , 791-803 (1993)). The interaction between Lex-Smad2 fragment and B42-p38 was analyzed by viability in yeast medium containing X-gal (Rho, SB et al., Proc Natl Acad Sci USA 96, 4488-93. (1999)) .

Construct cells that reliably produce p38DX2

Wild-type MEFs were transformed with pcNDA-p38DX2 or pcDNA and transformants were selected in DMEM medium containing 400 ug / ml G418. After removal of untransformed cells, the transformants were incubated in medium without G418 for 3 days, then fixed with 2% PFA and stained with Giemsa.

Immunostaining and Histological Analysis

Frozen tissue slides were fixed with 2% paraformaldehyde and washed three times with cold PBS. After blocking and permeablization with PBS containing 0.2% Triton X 100 (PBST) and 1% BSA, slides were incubated with anti-p38 antibody for 2 hours. After washing with PBS, incubated with anti-rabbit goat IgG-FITC and propidium iodide (PI 50 μg / ml) for 1 hour, then washed with PBS, fixed and confocal microscopy Was observed.

Construction of p38-DX2 Expression Vector

To construct the plasmid expressing p38-DX2, the cDNA of p38-DX2 was cloned into pcDNA3.1-myc. First, p38-DX2 was amplified using primers with EcoR1 and Xho I linker in H322 cDNA, and then cloned into pcDNA3.1-myc using EcoR1 and Xho 1. The pcDNA3-myc / p38DX2 vector expressing p38DX (FIG. 12) is Escherichia coli DH5alpha / p38-DX2. Deposited with accession number KCTC 10710BP as of date.

Lung cancer formation

32 p38 ^+/− mice (19 males and 13 females) and 25 p38 ^{+ / +} mice (14 males and 11 females) were used in the experiment. Mouse intraperitoneally received a single injection of benzopyrene (100 mg / kg). As a control, 3 p38 ^{+ / +} mice (2 males and 1 female) and 5 p38 ^+/− mice (4 males and 1 female) were placed in a beacon solution (10% DMSO and 35% PEG 40 in saline). This melted solution). Mice were slaughtered at the indicated times to fix lung tissue with formaldehyde, followed by H & E staining by the method of Kim, MJ et al., Nat. Genet. 34, 330-336, 2003 and observed with an optical microscope.

Example 1 Functional Significance and Mode of Action of p38 in TGF-β Signaling

In order to examine the important role of p38 in TGF-β signaling, p38 ^{+ / +} and p38– on TGF-β induced cell growth arrest, nuclear migration of Smad2 / 3, and the interaction of Smad2 / 3 with p38 /-The reaction of MEFs was investigated. Thymidine insertion, colony formation, and flow cytometry showed that growth of wild type cells was inhibited by TGF-β, whereas p38-deficient cells did not respond to these signals (each 1a, b and c). When MEFs were treated with TGF-β, Smad2 and Smad3 migrated to the nucleus in normal cells but not in p38 − / − cells (FIG. 1D). All these results indicate the functional significance of p38 in TGF-β signaling via Smad2 and 3. Possible interactions of p38 with Smad2 and 3 were investigated by co-immunoprecipitation. p38 interacted with Smad2 / 3, which was enhanced by TGF-β (FIG. 1E). Direct interaction between p38 and the two R-Smads was confirmed by East-to-hybrid and in vitro pull-down assays (FIG. 2, FIG. 7). The amount of p38 binding to Smad2 / 3 increased as p38 was induced by TGF-β (FIG. 2A). When p38 levels were increased by transformation, expression of TGF-β target genes was enhanced, indicating a stimulatory role of p38 in TGF-β signaling (FIG. 2B). Because p38 binds to both Smad2 and 3, we could predict that these two R-Smads would work in a similar fashion, so we looked more closely at the interaction between p38 and Smad2. Phosphorylation of TGF-β dependent Smad2 was inhibited in p38 ^- deficient MEFs, but recovered when p38 entered p38 ^{− / −} cells (FIG. 2C). The domains of Smad2 involved in interacting with p38 were examined by East to Hybrid (FIG. 2D) and in vitro pull-down assay (FIG. 7). In both experiments, the MH2 domain of Smad2 was found to be involved in the interaction.

To examine the mode of action of p38 in TGF-β signaling, we examined the TGF-β-induced binding of Smad2 and TGF-β receptors in normal and p38 deficient cells. Cells were treated with TGF-β and binding of Smad2 and receptors was monitored at time intervals by co-immunoprecipitation of Smad2 and type I receptors. In wild type cells, binding of TGF-β-induced Smad2 to TGF-β receptors was observed and decreased at an early time point, whereas in p38-deficient cells, Smad2-bound receptors accumulate later in TGF-β treatment. (FIG. 2E). In phosphorylation of TGF-β-induced Smad2, phosphorylated Smad2 gradually increased in wild-type cells, but was severely inhibited in P38 ^{− / −} cells (FIG. 2F). These results indicate that p38 plays a critical role in phosphorylation of TGF-β induced R-Smad through direct interaction with R-Smad2.

Example 2-Inhibition of p38 and Generation of Deletion Forms of p38 in Cancer Cells

To investigate the relationship between p38 and cancer formation, we examined the expression level of p38 in several different cancer cell lines. Three of the six cell lines examined showed low p38 levels in Western blot (FIG. 3A) and FACS analysis (FIG. 3B). The fact that all cell lines with low p38 levels expressed TGF-β type II receptors at normal levels and also retained kinase activity means that low p38 levels are not the result of receptor dysfunction. To see if the p38 level difference was due to transcriptional differences, RT-PCR was performed using different combinations of p38 specific primers. The p38 gene consists of four exons (FIG. 8A). When using the primers of SEQ ID NOS: 3 and 4 generating p38 cDNA penetrating exons 3 and 4, no decrease in p38 transcription was observed in cells showing reduced p38 levels (FIG. 3C, line 1), which was low. This means that p38 levels are not the result of low transcription. When primers of SEQ ID NOs: 5 and 6, which generate transcripts from exons 1 to 3, were used, small transcripts were obtained as well as expected transcripts (Fig. 3c, second line). Sequencing of the transcripts of the small size revealed that exon 2, which encodes 69 amino acids of p38, was deleted (FIG. 8B). In order to reconfirm the production of this small sized transcript, a primer of SEQ ID NO: 8 (FIG. 8B, primer DX-B) targeting the conjugation sequences of exon 1 and exon 3 generated by exon 2 deletion was made, and sequence number RT-PCR was performed using 7 primers together. Cell lines producing low levels of p38 produced small sized transcripts (named DX2, FIG. 3C, second and third lines).

Western blot analysis with an anti-p38 antibody generally means that only full-length p38 is detected and no DX2 is detected (FIG. 2A), which means that DX2 at the protein level is very unstable. In addition, low p38 levels in H460, H322 and H290 were demonstrated by immunofluorescence staining (FIG. 3D). To rule out the possibility of staining differences, H460 and DU145 cells were co-cultured on the same plate and stained p38. The staining intensity of p38 at H460 was much weaker than DU145 (FIG. 9A). No nuclear migration of TGF-β-dependent p38 was observed in cells showing low p38 levels (FIG. 3D, bottom row). To examine the association between p38 and TGF-β function, growth inhibition by TGF-β in the cell line was measured. Growth of A549 and DU145 cells was inhibited by TGF-β, whereas cells showing low p38 levels did not respond to TGF-β (FIG. 3E). This result is consistent with previous studies that A549 cells are sensitive to TGF-β and H460 cells are insensitive to TGF-β (Osada, H. et al. Cancer Res. 61, 8331-8339,2001; Kim, TK et al., Lung Cancer 31, 181-191, 2001). In addition, target genes in A549 and DU145 cells were induced by TGF-β but not in H322 and the other two cell lines showing low p38 levels (FIG. 3F, no data).

Example 3 Heterodimer Formation of Functional p38 with Inactive Deletion Mutants

To understand the general association between DX2 production and p38 inhibition, changes in p38 levels were investigated after DX2 transformation into DU145 cells. Western blot (FIG. 4A, first line) and FACS analysis (FIG. 9B) showed a decrease in p38 in DX2 transformed cells. In addition, the expression of c-myc, a target of p38, was increased by DX2 influx (FIG. 4A, line 3). DX2 alleviated growth arrest in response to TGF-β (FIG. 4B, FIG. 9C).

We discussed how DX2 affects functional p38 (p38-F). p38 can form homodimers (Quevillon, S. et al., J. Mol. Biol. 285, 183-195, 1999; Kim, JY et al., Proc. Natl. Acad. Sci. USA 99, 7912-7916, 2002), whether p38-DX2 interacts with p38-F was examined by an east-to-hybrid assay. p38-DX2 interacted with p38-F, but p38-DX did not interact with each other (FIG. 4C). In an in vitro pull-down assay, radiologically synthesized p38-F and -DX2 were co-purified with GST-p38-F (FIG. 4D), indicating that the direct interaction between p38-F and -DX2 To prove that it is. In order to determine whether DX2 is active in TGF-β signaling, we examined the interaction with Smad2 in an yeast-to-hybrid assay. p38 interacts with Smad2 as well as FBP known as the target of p38 (Kim, MJ et al., Nat. Genet. 34, 330-336, 2003), whereas DX2 did not bind at all with this protein (FIG. 4E). ).

To see how heterodimer formation inhibits p38, it was checked that p38 levels were regulated by proteasome-dependent degradation processes. DX2-producing H322 cells were treated with the prothiasome inhibitor ALLN (Zhou, M., et al., J. Biol. Chem. 271, 24769-24775, 1996) and p38 levels were increased by proteasome blocking. I looked at it. As can be seen in Western blot (FIG. 4F), immunofluorescence staining (FIG. 4G) and flow cytometry (FIG. 9D), p38 was markedly increased by ALLN treatment, which resulted in proteasome-mediated degradation of intracellular p38 levels. It is controlled by. In addition, p38-DX2 morphology was also detected by proteosome inhibition (FIG. 4F), confirming the notion that DX2 is unstable due to rapid proteosome-dependent degradation. The low intensity of p38-DX2 is due to low expression and / or insufficient recognition by anti-p38 antibodies as compared to normal p38 as demonstrated by RT-PCR analysis (FIG. 3C). Since p38 degradation is mediated by proteasomes, we examined whether ubiquitination was promoted by DX2. When immunoprecipitated p38 from control and DX2-transformed cells treated with ALLN and blotted with anti-ubiquitin antibody, higher amounts of ubiquitinated p38 compared to control cells in DX2-transformed cells Was observed (FIG. 4H). From the above, DX2 is believed to act as a dominant negative mutant that binds to p38 and forms an inert heterodimer that proceeds rapidly to the degradation process.

Example 4 Inactivation of TGF-β Signaling and Promoting Cell Growth by p38-DX2

After tracking the DX2 with MEFs, the effect of DX2 on cell growth was observed by microscopic analysis and colony formation. Cell growth was markedly increased by DX2 influx (FIG. 5A). In addition, three types of siRNA (si-DX2) capable of specifically binding to DX2 (FIG. 5B) were prepared, respectively, and transformed to H322 and H460 cells that produced DX2, and then transformed into normal p38 and The effect on the phosphorylation of smad2 was examined (FIG. 5C). When transforming H322 cells with 4 or 5 siRNA-expressing vectors, cell death by TGF-β was increased (left) and cell cycle arrest (right) occurred. 5d). A vector expressing siRNA 4 (si-DX2) that specifically inhibits the transcription of DX2 is introduced into H322 cells expressing DX2, and the siRNA checks whether normal levels of p38 and TGF-β signaling are restored. It was. si-DX2 eliminates DX2 transcripts (on FIG. 5E), normal p38 levels and phosphorylation of TGF-β-induced Smad2 (under FIG. 5E), nuclear transfer of Smad2 (FIG. 5F), TGF-β-dependent reporter Expression (FIG. 5G) and growth arrest (FIG. 5H) were restored. All of these results demonstrate those disruptive effects of DX2 on TGF-β signaling and cell growth regulation.

Example 5 Association of p38 Deletion Variants with Human Lung Cancer

Reduction of p38 is frequently found in various cancer cell lines (FIGS. 3A, b and d), and loss of p38 leads to over-proliferation of lung cells (Kim, MJ et al., Nat. Genet. 34, 330- 336, 2003) We looked at whether p38 abnormality is associated with lung cancer formation. To examine this possibility, chemical carcinogens, BP (benzo- (α) -pyrene) (Wang, Y. et al., Cancer Res. 63 , 4389-4395 (2003)), were identified as p38 ^{+ / +} and p38 ^{+. /} -Mouse intraperitoneally administered to induce lung tumors, the tumor formation in the lungs was examined. From 6 weeks after BP administration, lung tumors were observed at a frequency of 50-70% in p38 ^+/− mice and about 30% in normal mice (FIG. 6A), indicating that heterozygous mice It is more sensitive to BP-induced tumorigenesis. In situ immunofluorescence analysis using anti-p38 antibody showed a significant decrease in p38 at the tumor site (FIG. 6B). The lungs of BP-injected mice were examined for DX2 production. Tumors were generated in three of four lungs isolated from p38 ^+/− mice, whereas only one in three p38 ^{+ / +} mice developed tumors. All these tumors produced DX-2 (FIG. 10A), which supports the association of lung cancer formation with DX2.

P38 levels were compared in the inflammatory but non-tumor tumor tissues of the human lung. p38 was significantly reduced at the tumor site compared to the non-tumor site (FIG. 6C). DX2 was generated only in tumor tissue (FIG. 6D). RT-PCR and immunogenicity analyzes were performed on normal and tumor tissues isolated from the same patient to rule out the possibility of different p38 levels and DX2 formation in different individuals. Again, cancer-specific reduction of p38 was observed with high frequency (FIGS. 6E and 10B) and was associated with the production of DX2 (FIG. 6F). No DX2 was observed in one case showing normal p38 levels at the cancer site (FIGS. 6E and F, patient 22872). When pathologically examined 10 different samples diagnosed as lung adenocarcinoma, squamous cell carcinoma and large cell adenocarcinoma, cancer-specific reduction of p38 in 8 cases and DX2 The production of was observed (Table 1). In two cases, DX2 was found histologically in the normal region, but its occurrence was associated with low levels of p38.

(+) and (-) in the p38-F column refers to immunofluorescence staining of p38 determined by histological analysis in normal (N) and tumor (T) tissues. Positive and negative in the p38-DX2 column indicate the generation of DX2 measured by RT-PCR. Abbreviations are as follows. Recur (recurrence), SQC (squamous cell carcinoma), ADC (adenocarcinoma), LAC (large cell adenocarcinoma), Opdate (operation date)

Example 6 Association of Liver Cancer with p38, p38-DX2

We have investigated the association of p38 and p38-DX2 with liver cancer in human tissues.

In two hepatocellular carcinoma samples, p38-DX2 production was observed in normal and cancer tissues by RT-PCR. Formation of p38DX2 was observed in cancer tissues (FIG. 11A). In addition, the level of p38 was very low in the same liver cancer tissue (FIG. 11B).

Using p38-DX2 (protein, mRNA) specifically expressed in lung cancer or liver cancer of the present invention as a diagnostic marker for lung cancer or liver cancer, cancer can be accurately and easily diagnosed, and antibodies to p38-DX2 protein, siRNA, Inhibitors of p38-DX2 such as antisense nucleic acids and the like can be used to effectively prevent or treat lung or liver cancer.

<110> Seoul National University Industry Foundation <120> p38-DX2 and its use <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 756 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (753) P38DX2 amino acid sequence <400> 1 atg ccg atg tac cag gta aag ccc tat cac ggg ggc ggc gcg cct ctc 48 Met Pro Met Tyr Gln Val Lys Pro Tyr His Gly Gly Gly Ala Pro Leu   1 5 10 15 cgt gtg gag ctt ccc acc tgc atg tac cgg ctc ccc aac gtg cac ggc 96 Arg Val Glu Leu Pro Thr Cys Met Tyr Arg Leu Pro Asn Val His Gly              20 25 30 agg agc tac ggc cca gcg ccg ggc gct ggc cac gtg cag gat tac ggg 144 Arg Ser Tyr Gly Pro Ala Pro Gly Ala Gly His Val Gln Asp Tyr Gly          35 40 45 gcg ctg aaa gac atc gtg atc aac gca aac ccg gcc tcc cct ccc ctc 192 Ala Leu Lys Asp Ile Val Ile Asn Ala Asn Pro Ala Ser Pro Pro Leu      50 55 60 tcc ctg ctt gtg ctg cac agg ctg ctc tgt gag cac ttc agg gtc ctg 240 Ser Leu Leu Val Leu His Arg Leu Leu Cys Glu His Phe Arg Val Leu  65 70 75 80 tcc acg gtg cac acg cac tcc tcg gtc aag agc gtg cct gaa aac ctt 288 Ser Thr Val His Thr His Ser Ser Val Lys Ser Val Pro Glu Asn Leu                  85 90 95 ctc aag tgc ttt gga gaa cag aat aaa aaa cag ccc cgc caa gac tat 336 Leu Lys Cys Phe Gly Glu Gln Asn Lys Lys Gln Pro Arg Gln Asp Tyr             100 105 110 cag ctg gga ttc act tta att tgg aag aat gtg ccg aag acg cag atg 384 Gln Leu Gly Phe Thr Leu Ile Trp Lys Asn Val Pro Lys Thr Gln Met         115 120 125 aaa ttc agc atc cag acg atg tgc ccc atc gaa ggc gaa ggg aac att 432 Lys Phe Ser Ile Gln Thr Met Cys Pro Ile Glu Gly Glu Gly Asn Ile     130 135 140 gca cgt ttc ttg ttc tct ctg ttt ggc cag aag cat aat gct gtc aac 480 Ala Arg Phe Leu Phe Ser Leu Phe Gly Gln Lys His Asn Ala Val Asn 145 150 155 160 gca acc ctt ata gat agc tgg gta gat att gcg att ttt cag tta aaa 528 Ala Thr Leu Ile Asp Ser Trp Val Asp Ile Ala Ile Phe Gln Leu Lys                 165 170 175 gag gga agc agt aaa gaa aaa gcc gct gtt ttc cgc tcc atg aac tct 576 Glu Gly Ser Ser Lys Glu Lys Ala Ala Val Phe Arg Ser Met Asn Ser             180 185 190 gct ctt ggg aag agc cct tgg ctc gct ggg aat gaa ctc acc gta gca 624 Ala Leu Gly Lys Ser Pro Trp Leu Ala Gly Asn Glu Leu Thr Val Ala         195 200 205 gac gtg gtg ctg tgg tct gta ctc cag cag atc gga ggc tgc agt gtg 672 Asp Val Val Leu Trp Ser Val Leu Gln Gln Ile Gly Gly Cys Ser Val     210 215 220 aca gtg cca gcc aat gtg cag agg tgg atg agg tct tgt gaa aac ctg 720 Thr Val Pro Ala Asn Val Gln Arg Trp Met Arg Ser Cys Glu Asn Leu 225 230 235 240 gct cct ttt aac acg gcc ctc aag ctc ctt aag tga 756 Ala Pro Phe Asn Thr Ala Leu Lys Leu Leu Lys                 245 250 <210> 2 <211> 251 <212> PRT <213> Homo sapiens <400> 2 Met Pro Met Tyr Gln Val Lys Pro Tyr His Gly Gly Gly Ala Pro Leu   1 5 10 15 Arg Val Glu Leu Pro Thr Cys Met Tyr Arg Leu Pro Asn Val His Gly              20 25 30 Arg Ser Tyr Gly Pro Ala Pro Gly Ala Gly His Val Gln Asp Tyr Gly          35 40 45 Ala Leu Lys Asp Ile Val Ile Asn Ala Asn Pro Ala Ser Pro Pro Leu      50 55 60 Ser Leu Leu Val Leu His Arg Leu Leu Cys Glu His Phe Arg Val Leu  65 70 75 80 Ser Thr Val His Thr His Ser Ser Val Lys Ser Val Pro Glu Asn Leu                  85 90 95 Leu Lys Cys Phe Gly Glu Gln Asn Lys Lys Gln Pro Arg Gln Asp Tyr             100 105 110 Gln Leu Gly Phe Thr Leu Ile Trp Lys Asn Val Pro Lys Thr Gln Met         115 120 125 Lys Phe Ser Ile Gln Thr Met Cys Pro Ile Glu Gly Glu Gly Asn Ile     130 135 140 Ala Arg Phe Leu Phe Ser Leu Phe Gly Gln Lys His Asn Ala Val Asn 145 150 155 160 Ala Thr Leu Ile Asp Ser Trp Val Asp Ile Ala Ile Phe Gln Leu Lys                 165 170 175 Glu Gly Ser Ser Lys Glu Lys Ala Ala Val Phe Arg Ser Met Asn Ser             180 185 190 Ala Leu Gly Lys Ser Pro Trp Leu Ala Gly Asn Glu Leu Thr Val Ala         195 200 205 Asp Val Val Leu Trp Ser Val Leu Gln Gln Ile Gly Gly Cys Ser Val     210 215 220 Thr Val Pro Ala Asn Val Gln Arg Trp Met Arg Ser Cys Glu Asn Leu 225 230 235 240 Ala Pro Phe Asn Thr Ala Leu Lys Leu Leu Lys                 245 250 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for exon 3-4 <400> 3 agtgctttgg agaacagaat 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for exon 3-4 <400> 4 aagagcagag ttcatggagc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for exon 1-3 <400> 5 tctgacggtt tctgagcgtt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for exon 1-3 <400> 6 aagtgaatcc cagctgatag 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer for DX2 <400> 7 tgctttggtt ctgccatgcc g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer for DX2 <400> 8 cgtaatcctg cacgtggcca g 21 <210> 9 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> si-DX2 # 3 coding nucleic acid sequence <400> 9 tcgaggccac gtgcaggtta cgggagtagg ccgtaatcct gcacgtggcc tttt 54 <210> 10 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> si-DX2 # 3 coding nucleic acid sequence <400> 10 ctagaaaagg ccacgtgcag gattacggca gactcccgta atcctgcacg tggcc 55 <210> 11 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> si-DX2 # 4 coding nucleic acid sequence <400> 11 tcgagctggc cacgtgcagg attacgagta ctggtaatcc tgcacgtggc cagctttt 58 <210> 12 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> si-DX2 # 4 coding nucleic acid sequence <400> 12 ctagaaaagc tggccacgtg caggattacc agtactcgta atcctgcacg tggccagc 58 <210> 13 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> si-DX2 # 5 coding nucleic acid sequence <400> 13 tcgacacgtg caggattacg gggcgagtac tggccccgta atcctgcacg tgtttt 56 <210> 14 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> si-DX2 # 5 coding nucleic acid sequence <400> 14 ctagaaaaca cgtgcaggat tacggggcca gtactcgccc cgtaatcctg cacgtg 56 <210> 15 <211> 15 <212> DNA <213> Artificial Sequence <220> RT primer <400> 15 cagcaccacg tctgc 15  

Claims (25)

p38-DX2 protein, wherein the region of exon 2 of the p38 protein is deleted. The p38-DX2 protein of claim 1, having the amino acid sequence of SEQ ID NO: 2. 8. The p38-DX2 protein according to claim 1, which is a diagnostic marker for lung cancer or liver cancer. A nucleic acid molecule encoding the p38-DX2 protein of claim 1. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule of SEQ ID NO: 1. A recombinant vector comprising a nucleic acid molecule encoding the p38-DX2 protein of claim 1. A transformant transformed with the recombinant vector of claim 6. A method of preparing the p38-DX2 protein of claim 1 by culturing the transformant of claim 7. An antibody specific for the p38-DX2 protein of claim 1. Lung cancer or liver cancer diagnostic kit comprising an antibody specific for p38-DX2 protein of claim 1. Lung cancer or liver cancer diagnosis, comprising contacting the antibody specific for the p38-DX2 protein of claim 1 with a detection sample, forming an antigen-antibody complex, and comparing the amount of antigen-antibody complex formation with a control Method for detecting the marker protein p38-DX2. A siRNA nucleic acid molecule specific for mRNA of p38-DX2 protein of claim 1. 13. The siRNA nucleic acid molecule of claim 12, which is a sense and antisense RNA double chain encoded by the nucleotide sequences of SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, or SEQ ID NOs: 13 and 14. A pharmaceutical composition for treating lung cancer or liver cancer comprising the siRNA nucleic acid molecule of claim 12. A method of treating lung cancer or liver cancer by administering the siRNA nucleic acid molecule of claim 12. An antisense nucleic acid molecule having a sequence complementary to the mRNA of the p38-DX2 protein of claim 1. A pharmaceutical composition for treating lung cancer or liver cancer comprising the antisense nucleic acid molecule of claim 16. A method of treating lung cancer or liver cancer by administering the antisense nucleic acid molecule of claim 16. A method for detecting the mRNA of the p38-DX2 lung cancer diagnostic marker protein p38-DX2 using a primer or probe that distinguishes the mRNA of the p38-DX2 protein and the mRNA of the p38 protein of claim 1. The method of claim 19, wherein the primers are SEQ ID NOs: 5 and 6, or SEQ ID NOs: 7 and 8. The method of claim 19, wherein the probe is SEQ ID NO: 8. A method for screening an inhibitor that inhibits heterodimer formation of p38-DX2 protein and p38 protein of claim 1. The method of claim 22, wherein the method is screened by a yeast-two-hybrid or in vitro pull-down assay. The expression of the p38-DX2 protein of claim 1 comprising culturing a cell expressing the p38-DX2 protein of claim 1, treating the candidate inhibitor to the cell and comparing mRNA or protein levels with the control. A method of screening inhibitors for inhibition. The method of claim 24, screened by reverse transcription-polymerase chain reaction (RT-PCR).

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