Patent application title: p53-DEPENDENT APOPTOSIS-INDUCING PROTEIN AND METHOD OF SCREENING FOR APOPTOSIS REGULATOR
Yusuke Nakamura (Yokohama-Shi, JP)
Hirofumi Arakawa (Tokyo, JP)
Japan as Represented be the President of the University of Tokyo
Oncotherapy Science, Inc.
IPC8 Class: AG01N33574FI
Class name: Involving a micro-organism or cell membrane bound antigen or cell membrane bound receptor or cell membrane bound antibody or microbial lysate animal cell tumor cell or cancer cell
Publication date: 2008-09-11
Patent application number: 20080220455
p53-dependent Damage-Inducible Nuclear Protein 1 (p53DINP1 protein) is a
p53-induced nuclear protein that induces p53-dependent apoptosis by
regulating p53 function through Ser 46 phosphorylation. A DNA encoding
p53DINP1 can be applied as anticancer agents for destroying neoplasms
such as tumors, and as therapeutic or preventive agents for diseases
associated with p53-mediated apoptosis abnormalities. It is also possible
to apply the above protein and DNA in methods of screening for candidate
compounds for regulating p53-mediated apoptosis.
1. An isolated DNA of the following (a) or (b):(a) a DNA encoding a
protein comprising the amino acid sequence of SEQ ID NOs: 2 or 4, or(b) a
DNA comprising the coding region of a nucleotide sequence of any one of
SEQ ID NOs: 1, 3, and 5.
2. An isolated DNA of the following (a) or (b) encoding a protein having the activity to induce apoptosis:(a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NOs: 2 or 4, wherein one or more amino acids are substituted, deleted, inserted, and/or added, or(b) a DNA hybridizing under stringent conditions to a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5.
3. A DNA containing at least 15 nucleotides, wherein said DNA is complementary to a DNA comprising a nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5, or to the complementary strand thereof.
4. An isolated protein encoded by the DNA of claim 1 or 2.
5. A vector comprising the DNA of claim 1 or 2.
6. A host cell carrying the DNA of claim 1 or 2, or the vector of claim 5.
7. A method for producing the protein of claim 4, wherein said method comprises the steps of culturing the host cell of claim 6, and recovering the protein expressed by said host cell.
8. An antibody binding to the protein of claim 4.
9. An antisense polynucleotide to the DNA comprising a nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5.
10. A method of screening for a candidate compound for an apoptosis-regulating agent, wherein said method comprises the steps of:(a) contacting a test sample with the protein of claim 4,(b) detecting the binding activity of the test sample to said protein, and(c) selecting a compound having the activity to bind to said protein.
11. A method of screening for a candidate compound for an apoptosis-regulating agent, wherein said method comprises the steps of:(a) contacting a test sample with the protein of claim 4 in the presence of p53,(b) measuring the phosphorylation level of said p53 protein at the Ser46 residue after the contact, and(c) selecting a compound capable of regulating apoptosis based on the phosphorylation level measured in step (b).
12. A method of screening for a candidate compound for an apoptosis-regulating agent, wherein said method comprises the steps of:(a) contacting a test sample with a cell containing a vector having the structure in which the p53-binding sequence of SEQ ID No: 8 and a reporter gene are operably linked, or an extract of said cell,(b) measuring the expression level of said reporter gene, and(c) selecting a compound capable of decreasing or increasing the expression level of said reporter gene measured in step (b), compared to that measured in the absence of the test sample.
13. An apoptosis-regulating agent comprising as an effective ingredient a compound selected by a method of any one of claims 10 to 12.
CROSS-REFERENCES TO RELATED APPLICATIONS
The present patent application is a continuation application of U.S. patent application Ser. No. 10/484,157, filed Jul. 26, 2004, which is a National Stage Entry of PCT/JP2002/07305, filed Jul. 18, 2002, which claims the benefit of JP 2001-220349, filed Jul. 19, 2001, the disclosures of which are hereby incorporated herein by reference in their entirety for all purposes.
The present invention relates to a novel p53-dependent apoptosis-inducing protein, a gene that encodes the protein, and such. It also relates to a method of screening for compounds capable of regulating p53-dependent apoptosis.
Of the genes known to be involved in human cancer, mutations have been most commonly detected in the tumor suppressor gene p53. The p53 gene product activates transcription of many downstream genes, and by regulating their transcription, exerts a variety of biological functions. The two main functions of p53 are cell cycle arrest and apoptosis, mediated by p21.sup.waf1 and BAX respectively. These functions were thought to be characteristics essential for p53-dependent tumor suppression (el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993). WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817-825; Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993). The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805-816; Miyashita, T. and Reed, J. C. (1995). Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293-299).
The present inventors recently discovered p53R2, a novel p53 target molecule that supplies deoxyribonucleotides for DNA repair. Thus, p53 is also directly involved in DNA repair (Tanaka, H., Arakawa, H., Yamaguchi, T., Shiraishi, K., Fukuda, S., Matsui, K., Takei, Y., and Nakamura, Y. (2000). A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 404, 42-49).
To date, it has been reported that a large number of p53 target genes, and approximately one hundred candidates for p53-binding sequences, are present in human chromosomes. It is thought the bioactivity of this vast number of p53 target genes is reflected in p53's tumor suppressing actions resulting from by p53-mediated cell cycle arrest, DNA repair and apoptosis. Therefore, identifying additional p53 target genes is important in elucidating the mechanisms of tumorigenesis, and cell protection from genotoxic stresses.
Induction of apoptosis is known to be the most important of p53's tumor suppressing functions, and this activity is being used to kill cancer cells in human patients (Levine, Cell: 88, 323-331, 1997). However, despite this kind of evidence, the mechanism of p53-mediated apoptosis has yet to be elucidated.
BAX, Fas, Killer/DR5, and PIGs are recognized as candidates for mediating p53-dependent apoptosis, and their functions are currently being investigated. However, acting alone, any one of these candidates is insufficient to induce apoptosis. The current inventors have found p53AIP1, a novel target gene for p53, which differs from the candidates described above in that it can induce apoptosis independently when over-expressed in some cancer cells. Blocking p53AIP1 expression inhibits p53-mediated apoptosis (Oda, K., Arakawa, H., Tanaka, T., Matsuda, K., Tanikawa, C., Mori, T., Nishimori, H., Tamai, K., Tokino, T., Nakamura, Y., and Taya, Y. (2000) p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 102, 849-862). These results suggested that p53AIP1 was a crucial mediator in the mechanism of p53-dependent apoptosis.
Growing evidence suggests that the biological activity of p53 is determined by modifications such as phosphorylation, acetylation, and the like (Giaccia, A. J. and Kastan, M. B. (1998). The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev. 19, 2973-2983). For example, there are reports indicating that phosphorylation of the p53 protein at its Ser15 and Ser20 residues is important in p53 activation in response to DNA damage (Shieh, S. Y., Ikeda, M., Taya, Y., and Prives, C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325-334; Shieh, S. Y., Taya, Y., and Prives, C. (1999) DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, Ser20, requires tetramerization. EMBO J. 18, 1815-1823). There are also reports that acetylation of the p53 C-terminal domain enhances DNA binding activity (Gu, W. and Roeder, R. G. (1997). Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595-606). In addition, ATM and CHK2 proteins have been proposed as strong candidates for a kinase involved in the phosphorylation of p53 at Ser15 and Ser20 respectively (Sarkaria, J. N., Busby, E. C., Tibbetts, R. S., Roos, P., Taya, Y., Karnitz, L. M, and Abraham, R. T. (1999). Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res. 59, 4375-4382; Hirao, A., Kong, Y. Y., Matsuoka, S., Wakeham, A., Ruland, J., Yoshida, H., Liu, D., Elledge, S. J., and Mak, T. W. (2000). DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science 287, 1824-1827). Furthermore, it has been shown that phosphorylation of the Ser46 residue is essential for p53-induced apoptosis (Oda et al., supra).
Based on the various findings described above, the present inventors speculated that p53 determines whether cells with damaged DNA will survive or be killed, such that cells which are extremely damaged or have been exposed to danger are eliminated through phosphorylation of p53 at Ser46, and induction of p53AIP1.
Because known p53 targets do not sufficiently explain this speculation, the present inventors used a method capable of directly cloning human chromosome-derived p53-binding sequences for further screening of p53 targets. The present inventors identified four novel target genes and a GPI-anchored molecule-like protein (GML), whose expressions are induced by wild-type p53 (Furuhata, T., Tokino, T., Urano, T., and Nakamura, Y. (1996). Isolation of a novel GPI-anchored gene specifically regulated by p53; correlation between its expression and anti-cancer drug sensitivity. Oncogene 13, 1965-1970; Kimura, Y., Furuhata, T., Urano, T., Hirata, K., Nakamura, Y., and Tokino, T. (1997). Genomic structure and chromosomal localization of GML (GPI-anchored molecule-like protein), a gene induced by p53. Genomics 41, 477-480), P2XM (Urano, T., Nishimori, H., Han, H., Furuhata, T., Kimura, Y., Nakamura, Y., and Tokino, T. (1997). Cloning of P2XM, a novel human P2X receptor gene regulated by p53. Cancer Res. 57, 3281-3287), BAI1 (Nishimori, H., Shiratsuchi, T., Urano, T., Kimura, Y., Kiyono, K., Tatsumi, K., Yoshida, S., Ono, M., Kuwano, M., Nakamura, Y., and Tokino, T. (1997). A novel brain-specific p53-target gene, BAIL, containing thrombospondin type 1 repeats inhibits experimental angiogenesis. Oncogene 15, 2145-2150) and CSR (Han, H. J., Tokino, T., and Nakamura, Y. (1998). CSR, a scavenger receptor-like protein with a protective role against cellular damage caused by UV irradiation and oxidative stress. Hum. Mol. Genet. 7, 1039-1046).
The present inventors isolated a p53-inducible transcript by establishing a cell line in which p53 expression is regulated under set conditions, and then applied differential display techniques to that cell line (Takei, Y., Ishikawa, S., Tokino, T., Muto, T., and Nakamura, Y. (1998). Isolation of a novel TP53 target gene from a colon cancer cell line carrying a highly-regulated wild-type TP53 expression system. Genes Chromosomes Cancer 23, 1-9). Using this approach, three additional novel p53 target genes were identified: TP53TG1 (Takei et al., supra), TP53TG3 (Ng, C. C., Koyama, K., Okamura, S., Kondoh, H., Takei, Y., and Nakamura, Y. (1999). Isolation and characterization of a novel TP53-inducible gene, TP53TG3. Genes Chromosomes Cancer 26, 329-335) and p53R2 (Tanaka, H., Arakawa, H., Yamaguchi, T., Shiraishi, K., Fukuda, S., Matsui, K., Takei, Y., and Nakamura, Y. (2000). A ribonucleotide reductase gene involved in p53-dependent cell-cycle checkpoint for DNA damage. Nature 404, 42-49). Genes already known to be activated or suppressed by p53 were also identified during this approach.
DISCLOSURE OF THE INVENTION
Although novel p53 target genes have been identified one after another, many of the genes involved in p53-induced apoptosis have yet to be identified. Among the many functions of p53, target genes which can prompt cells to apoptosis have yet to be elucidated. These p53 target genes are extremely significant because they are useful for 1) actively inducing cancer cell apoptosis, and 2) elucidating the mechanism by which p53 determines whether to kill cells or to let them live.
Therefore, an objective of the present invention is to provide a novel protein that induces p53-mediated apoptosis, and a gene encoding that protein. The present invention also provides a novel method of screening for a compound that regulates apoptosis.
To achieve the above-described objectives, the present inventors conducted exhaustive studies, and as a result, identified the p53-dependent Damage-Inducible Nuclear Protein 1 (the p53DINP1 protein). The p53DINP1 protein is a p53-induced nuclear protein that induces p53-dependent apoptosis by regulating p53 function through Ser 46 phosphorylation. A cDNA and a genomic DNA encoding p53DINP1 were also identified. The present inventors also developed an antisense oligonucleotide to p53DINP1, which regulates apoptosis, and a method of screening for a compound that regulates apoptosis, and such.
More specifically, the present invention relates to:
 an isolated DNA of the following (a) or (b):
(a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NOs: 2 or 4, or(b) a DNA comprising the coding region of a nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5;
 an isolated DNA of the following (a) or (b) encoding a protein having the activity to induce apoptosis:
(a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NOs: 2 or 4, wherein one or more amino acids are substituted, deleted, inserted, and/or added, or(b) a DNA hybridizing under stringent conditions to a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5;
 a DNA containing at least 15 nucleotides, wherein said DNA is complementary to a DNA comprising a nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5, or to the complementary strand thereof;
 an isolated protein encoded by the DNA of  or ;
 a vector comprising the DNA of  or ;
 a host cell carrying the DNA of  or , or the vector of ;
 a method for producing the protein of , wherein said method comprises the steps of culturing the host cell of , and recovering the protein expressed by said host cell;
 an antibody binding to the protein of ;
 an antisense polynucleotide to the DNA comprising a nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5;
 a method of screening for a candidate compound for an apoptosis-regulating agent, wherein said method comprises the steps of:
(a) contacting a test sample with the protein of ,(b) detecting the binding activity of the test sample to said protein, and(c) selecting a compound having the activity to bind to said protein;
 a method of screening for a candidate compound for an apoptosis-regulating agent, wherein said method comprises the steps of:
(a) contacting a test sample with the protein of  in the presence of p53,(b) measuring the phosphorylation level of said p53 protein at the Ser46 residue after the contact, and(c) selecting a compound capable of regulating apoptosis based on the phosphorylation level measured in step (b);
 a method of screening for a candidate compound for an apoptosis-regulating agent, wherein said method comprises the steps of:
(a) contacting a test sample with a cell containing a vector having the structure in which the p53-binding sequence of SEQ ID No: 8 and a reporter gene are operably linked, or an extract of said cell,(b) measuring the expression level of said reporter gene, and(c) selecting a compound capable of decreasing or increasing the expression level of said reporter gene measured in step (b), compared to that measured in the absence of the test sample; and
 an apoptosis-regulating agent comprising as an effective ingredient a compound selected by a method of any one of  to .
The present invention is described in detail below.
The present invention relates to a novel protein p53DINP1, whose expression is induced by p53. p53DINP1 is involved in the phosphorylation of p53 at the Ser46 residue during p53-induced apoptosis caused by DNA damage such as double-strand breaks. P53DINP1 also enhances expression of the p53-dependent apoptosis protein, p53AIP1.
Preferred examples of such a protein include "p53DINP1a" and "p53DINP1b", comprising amino acid sequences shown in SEQ ID NOs: 2 and 4 respectively (unless otherwise stated, "p53DINP1a" and "p53DINP1b" proteins are hereinafter collectively described as "p53DINP1" proteins). The present invention also includes proteins analogous to "p53DINP1" proteins, as long as their physiological activity is the same as described above. Such analogous proteins include mutant "p53DINP1" proteins produced artificially, or isolated from humans or other organisms, and having amino acid sequences with one or more amino acid substitutions, deletions, insertions, and/or additions compared with those shown in SEQ ID NOs: 2 or 4. Such analogous proteins also include proteins encoded by DNAs that hybridize to the cDNAs that encode p53DINP1.
As used herein, "isolated" refers to material (for example, a polynucleotide or a polypeptide) which has been extracted from its original environment (for example, the natural environment if it occurs naturally), and which has been altered "by means of human intervention". "Isolated" also refers to material present in a sample and substantially rich in a compound of interest, and/or material present in a sample containing the compound of interest in a partially or substantially purified form. As used herein, the term "substantially purified" refers to a compound that has been separated from its natural environment and is at least 60% or more, preferably 75% or more, and most preferably 90% or more free of other components with which the compound naturally occurs.
With regards to "amino acid sequences with one or more amino acid substitutions, deletions, insertions, and/or additions", the number or position of amino acid mutations is not limited, as long as mutants maintain p53DINP1 protein functions. The proportion of mutations in the protein is typically 10% or less, preferably 5% or less, and most preferably 1% or less of total amino acids.
The "p53DINP1" proteins can be prepared by extraction from the tissues of mammals, for example, of primates such as humans or rodents such as mice, using an anti-p53DINP1 antibody as described below, or the like. All human cells express p53DINP1 proteins, so the type of tissue is not limited as long as human tissue is used. The thymus, pancreas, spleen, testis, and peripheral leukocytes express large amounts of p53DINP1 protein and are thus suitable for use. A transformant comprising a vector carrying a DNA that encodes p53DINP1 can be conveniently used for p53DINP1 purification, as described below.
Proteins analogous to the above described p53DINP1 proteins can be prepared using a hybridization technique known to those skilled in the art. For example, by using a nucleotide sequence encoding p53DINP1 (for example, a nucleotide sequence from SEQ ID NOs: 1, 3, or 5) or a portion thereof as a probe, proteins analogous to the p53DINP1 proteins can be obtained by isolating, from a variety of mammals including humans and other organisms, DNAs highly homologous to p53DINP1 cDNA. DNAs highly homologous to p53DINP1 cDNA can also be prepared by PCR (polymerase chain reaction) using as a primer the nucleotide sequence shown in SEQ ID NOs: 1, 3, or 5, or a portion thereof. PCR is known to those skilled in the art.
Those skilled in the art can select stringent hybridization conditions appropriate for isolating DNAs encoding polypeptides functionally equivalent to the p53DINP1 polypeptides. For example, pre-hybridization is carried out in a hybridization solution containing 25% formamide (50% formamide under more stringent conditions), 4×SSC, 50 mM Hepes (pH 7.0), 10×Denhardt's solution, and 20 μg/ml denatured salmon sperm DNA at 42° C. overnight. A labeled probe is added and hybridization is carried out by incubation at 42° C. overnight. Post-hybridization washes are carried out under different levels of stringency including moderately stringent "1×SSC, 0.1% SDS, 37° C.", highly stringent "0.5×SSC, 0.1% SDS, 42° C.", and more highly stringent "0.2×SSC, 0.1% SDS, 65° C." conditions. As the stringency level of post-hybridization washes increases, DNA more highly homologous to the probe sequence is expected to be isolated. The above-described combinations of SSC, SDS, and temperature are merely examples of wash conditions. Those skilled in the art can achieve the same stringencies as those described above by appropriately combining the above factors or others (such as probe concentration, probe length, hybridization period, etc.) that affect hybridization stringency.
A polypeptide thus isolated using hybridization will usually comprise an amino acid sequence highly homologous to the polypeptides identified by the present inventors. "Highly homologous" refers to sequence homology of at least 40% or more, preferably 60% or more, further preferably 80% or more, further preferably 90% or more, further preferably at least 95% or more, further preferably at least 97% or more (for example, 98% to 99%). Amino acid sequence identity can be determined, for example, using the BLAST algorithm according to Karlin and Altschul (Proc. Natl. Acad. Sci. USA. 87:2264-2268, 1990; Proc. Natl. Acad. Sci. USA. 90: 5873-5877, 1993). A program designated BLASTX has been developed based on this algorithm (Altschul et al., J. Mol. Biol. 215: 403-410, 1990). For analysis of amino acid sequence identity, the BLASTX program uses, for example, a score of 50 and a word length of 3 as parameters. The default parameters of the BLAST and the Gapped BLAST programs respectively are used. Specific methodology for performing BLAST analyses is publicly available on the World Wide Web at ncbi.nlm.nih.gov.
Proteins structurally analogous to p53DINP1 can be prepared not only from naturally occurring proteins, but also by artificially modifying p53DINP1 proteins comprising a sequence shown in SEQ ID NO: 2 or 4. This kind of artificial modification of DNAs coding for p53DINP1 proteins, for example the DNA of SEQ ID NO: 1, 3, or 5, can be undertaken by those skilled in the art using known methods, including PCR, cassette mutagenesis, or site-directed mutagenesis using deletion-mutants.
To ascertain whether the p53DINP1-analogous proteins thus obtained comprise activities similar to p53DINP1, such as participation in p53 Ser46 phosphorylation and induction of p53AIP1 expression, Western blot analysis using antibodies specific to phosphorylated p53 Ser46 and p53AIP1 respectively, can be used.
The p53DINP1 proteins and their above-described analogous proteins induce apoptosis-related p53AIP1 expression, and also enhance p53 Ser46 phosphorylation, a process involved in the initial signal of the p53-induced apoptosis pathway. Thus these proteins are effective as pharmaceutical agents capable of inducing apoptosis of neoplasms such as tumors that are unfavorable to organisms. They can also be used for the production of antibodies against p53DINP1 proteins and such. To produce such antibodies, it is not always necessary to use the entire sequence of a protein comprising an above-mentioned activity. A portion of the protein can also be used.
The DNAs encoding the "p53DINP1" proteins of the present invention also include cDNA, genomic DNA and synthetic DNA, as long as the DNA encodes 1) a protein having the function of inducing p53 phosphorylation at the Ser46 residue after a double stranded-DNA break or the like, and/or 2) a protein comprising the activity of inducing p53AIP1 expression.
Preferable cDNAs that encode "p53DINP1" proteins comprise, for example, the sequence of SEQ ID NO: 1 or 3, but are not limited thereto. Favorable cDNAs also include those comprising the same function as p53DINP1 cDNA. Such cDNAs can be selected from a cDNA library derived from a tissue of an organism expressing a protein comprising an above-mentioned activity. This can be achieved by hybridization using a labeled DNA probe comprising the sequence of SEQ ID NO: 1 or 3, or a portion thereof. The above cDNAs can also be prepared by RT-PCR, using as a template, total RNA derived from a tissue of an organism that expresses a protein comprising an above-mentioned activity, and using as a primer a synthetic oligonucleotide that includes a portion of SEQ ID NO: 1 or 3.
One example of the preferred genomic DNAs encoding the p53DINP1 proteins comprises the sequence of SEQ ID NO: 5. Since this genomic DNA is mapped to human chromosome 8q22, it can be isolated from a genomic DNA library prepared using chromosomal DNA containing the 8q22 region. In addition to DNA comprising the sequence of SEQ ID NO: 5, genomic DNA encoding a protein comprising the above activity can also be isolated using the above tissue-derived genomic DNA library previously used for isolation of the above cDNA.
The above-described DNAs can also be synthesized using a commercially available DNA synthesizer. For example, DNAs having the sequence of SEQ ID NOs: 1, 3, or 5, or complementary strands thereof are synthesized, and these DNAs are annealed to prepare desired double stranded DNAs.
The above-mentioned DNAs encoding "p53DINP1" proteins, and in particular the cDNAs, are useful in the production of proteins comprising the activity of inducing p53 Ser46 phosphorylation, and the like. When used to produce such proteins, the DNAs are preferably inserted into an appropriate expression vector.
An appropriate expression vector can be suitably selected in accordance with the translation system used for protein production. A cellular or cell-free translation system can be selected, depending on the purpose. In a cellular translation system, vectors that can be used for expression in Escherichia coli include pkk223-3, pkk233-2, and pJLA502. A protein of the present invention can also be expressed as a fusion protein with other proteins. Vectors for fusion protein expression are exemplified by pRIT2T, pGEX-2T, pGEX-3×, and such. Fusion proteins are easily recovered using an affinity column. The use of a vector comprising a thrombin- or factor Xa-cleavage site at the connecting site of the fusion partners enables specific recovery of the target protein. Examples of vectors for secretion of proteins into the periplasm or outside of cells include pKT280 and pRIT5 (Okada, M. and Miyazaki, K., ed. Formidable Biotechnical Series. Protein Experimental Note, I, Extraction and Separation/Purification, Yodosha, 1996, pp. 139-149). The proteins of this invention may also be expressed in insect and mammalian cells using baculoviruses. An example of a baculovirus vector for use in mammalian cells is pAcCAGMCS1 (Muramatsu, M., ed. Laboratory Manual for Gene Technology, 3rd ed., Maruzen, 1996, pp. 242-246).
Recombinant proteins expressed in host cells can be purified by a method well known in the art. When a protein of this invention is expressed in the form of a fusion protein linked at the N-terminus to a histidine residue tag, glutathione-S-transferase (GST), or such, it can be purified through a nickel column, glutathione Sepharose column, etc.
DNAs encoding "p53DINP1" proteins of the present invention may be applied in gene therapy of disorders caused by their mutation or deletion. They may also be useful for apoptosis induction in unfavorable neoplasms such as tumors. When applied as above, it is preferable to insert the DNA into a vector to deliver that DNA into the desired tissue or cell. Examples of a vector used for gene therapy include viral vectors such as retroviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia virus vectors, lentivirus vectors, herpes virus vectors, alphavirus vectors, EB virus vectors, papillomavirus vectors, and foamy virus vectors; and non-viral vectors such as cationic liposomes, ligand DNA complexes, gene guns (Y. Niitsu et al., Molecular Medicine 35, 1385-1395, 1998), and such. Gene transduction may be carried out in vivo, ex vivo, etc.
The above-described DNA can be used as a full length DNA, or as a portion thereof, in hybridization probes, PCR primers, or ribozyme derivatives. Fragment length is preferably enough to ensure probe specificity and such, for example, a length of at least 15 nucleotides. Examples of such polynucleotides are those that specifically hybridize to DNAs that comprise the nucleotide sequence of SEQ ID NO: 1, 3, or 5, or to a complementary strand thereof. As used herein, the phrase "specifically hybridize" means that no significant cross-hybridization to DNAs encoding other proteins occurs during hybridization. When applied to cloning of the DNAs encoding the proteins of the present invention, or to restriction fragment length polymorphism (RFLP) analysis and such, the above-mentioned probes and primers can be used to detect polymorphisms or mutations in genes or cDNAs.
In addition to p53, p53-binding sequences included in the DNA of SEQ ID NO: 1, 3, or 5, for example, partial sequences containing the sequence of SEQ ID NO: 8, can be used to regulate transcription by binding to p53 and hence regulating expression of an operably-linked downstream gene.
The present invention also relates to antisense polynucleotides of DNAs that encode the proteins of this invention. The antisense polynucleotides of this invention suppress expression of the proteins of the present invention, and are hence useful in developing reagents for elucidating the mechanisms of disorders associated with p53-dependent apoptosis, and in developing drugs for the treatment of these disorders. Examples of such antisense polynucleotides are those having the sequences of SEQ ID NOs: 15 and 16. In addition, the antisense polynucleotides comprise those that hybridize to a nucleotide sequence of SEQ ID NO: 1 or 3, or to a nucleotide sequence within the coding region of SEQ ID NO: 5. Furthermore, the antisense polynucleotides need not be completely complementary to the nucleotide sequence of SEQ ID NO: 1 or 3, or to the nucleotide sequence within the coding region of SEQ ID NO: 5, as long as they are capable of effectively inhibiting the expression of the proteins of the present invention.
The present invention also relates to antibodies binding to the proteins of this invention. The antibodies of this invention include both polyclonal and monoclonal antibodies, as long as they can bind specifically to the proteins of this invention. The polyclonal antibodies are prepared according to the well-known method in which animals such as rabbits and guinea pigs are immunized with a protein of this invention, or a partial peptide thereof, the elevation of antibody titer is confirmed, and peripheral blood from the immunized animals is collected to obtain the antiserum. Monoclonal antibodies can be prepared according to a well known method in which animals such as mice are immunized with a protein of this invention or a partial peptide thereof, the elevation of antibody titer is confirmed, and the spleen (or lymph nodes) is collected from the immunized animals. Antibody-producing cells from the spleen or lymph nodes are then fused with myeloma cells to prepare hybridomas. Monoclonal antibodies can be prepared from the culture supernatant of the hybridoma producing those antibodies.
These antibodies can be used to detect expression levels and such of the proteins of this invention, and thus can be used for affinity purification of the proteins of this invention. They can also be used for testing and diagnosing disorders in test subjects, where these disorders are caused by an abnormality in expression or structure of a protein of this invention. For patients introduced with a DNA of this invention for the purposes of gene therapy and the like, expression of a protein of the present invention from the introduced DNA can be also monitored using these antibodies. The antibodies can also be used as a means of obtaining, by immunoprecipitation, a factor that interacts with a protein of this invention. In particular, the antibodies of this invention can be used to obtain a kinase that catalyses phosphorylation of p53 at the Ser46 residue, a process expected to signal p53-dependent apoptosis induction, since the proteins of this invention interact with this kinase. Methods such as ELISA and Western blotting can also be used as required for the detection of a protein of this invention.
The present invention also relates to methods of screening for candidate compounds for an apoptosis-regulating agent. The first screening method comprises the steps of: (a) contacting a test sample with a protein of this invention; (b) detecting the binding activity of the test sample to the protein or partial peptide thereof; and (c) selecting a compound comprising binding activity.
To give a specific example, a compound binding to a protein of the present invention is first screened by contacting with a test sample (for example, a culture supernatant or cell extract) expected to contain a compound capable of binding to the protein. An antibody of this invention is added, and the compound is then immunoprecipitated along with the protein of this invention. The binding of the candidate compound to the protein of this invention can be detected based, for example, on mobility shift during electrophoresis of the immunoprecipitated products. In addition, recovery of the candidate compound from a sample in which binding is detected can be carried out by using the binding activity to the protein of this invention, for example, by using affinity chromatography.
Screening may also be carried out using "West Western blotting" which comprises the steps of: (a) using a phage vector to prepare a cDNA library from tissues or cells that presumably express a protein that binds to a protein of this invention, (b) expressing the cDNA library on agarose, (c) transferring the protein onto a membrane, and (d) reacting it with the labeled protein of this invention to detect plaques that express the binding protein. Systems such as the "two-hybrid system" may also be used. In the "two-hybrid system", expression of a reporter gene in which a GAL4 DNA binding region and GAL4 transcription activation region are linked downstream a promoter comprising a binding sequence of a protein of this invention, is used to detect binding between the test protein and the protein of this invention.
Furthermore, methods well-known to those skilled in the art include 1) a method of screening for binding molecules by immobilizing a protein of the present invention on a solid phase or the like, and then reacting it with synthetic compounds, natural product banks, or random phage peptide display libraries, and 2) a method for isolating candidate compounds by high throughput screening using combinatorial chemistry.
Alternatively, since the proteins of this invention have the activities of inducing p53AIP1 expression and p53 Ser46 phosphorylation, candidate compounds that bind to the proteins of this invention can be screened and selected based on changes in these activities due to binding with the candidate compound. More specifically, a candidate compound, whose binding activity has been detected by a variety of methods as described above, can be also selected based on its effect (promotion or inhibition) on the activity of a protein of this invention. For example, p53 Ser46 phosphorylation can be measured by contacting candidate compounds with a protein of the present invention in the presence of p53, then using Western blotting, ELISA or such using an antibody that specifically recognizes the phosphorylated p53 protein at the Ser46 residue.
Examples of test samples for this screening include, but are not limited to, cell extracts, gene library expression products, synthetic low molecular weight compounds, proteins, natural or synthetic peptides, natural compounds, and sera. A compound that are isolated by the above-described screening, i.e., a screening using as an index the binding activity to a protein of this invention, may also be used as a test samples.
A protein of this invention comprises the functions of 1) inducing p53 phosphorylation at the Ser46 residue, a process thought to be the upstream signal for the induction of p53-dependent apoptosis caused by DNA damage such as double-strand breaks, or 2) inducing the expression of a "p53AIP1" protein in the induction of apoptosis. Thus it is possible that compounds selected by the screening methods of this invention can be used to regulate the action of p53 in switching to the apoptosis action amongst the variety of p53 actions, and to control p53-dependent apoptosis induction, a process mediated by the proteins of this invention. As used herein, "regulate" also includes "enhance", "inhibit" or "suppress". Therefore, an apoptosis-enhancing compound may be applied as an agent capable of actively inducing apoptosis of neoplasms such as tumors, and an apoptosis-inhibiting or suppressing compound may be applied as a preventative or therapeutic agent for disorders in p53DINP1-mediated apoptosis.
The present invention also relates to another method of screening candidate compounds for a second apoptosis-regulating agent. This screening method of this invention comprises the steps of: (a) contacting a test sample with a cell containing a vector having the structure in which the p53-binding sequence in the p53DINP1 genome sequence and a reporter gene are operably linked; (b) measuring the expression level of said reporter gene, and (c) selecting a compound capable of decreasing or increasing the expression level of said reporter gene measured in step (b), compared to that measured in the absence of the test sample.
The expression "p53-binding sequence" used herein refers to a sequence in the p53DINP1 genomic DNA to which p53 protein binds to induce transcription of a "p53DINP1" gene, a p53 protein target gene. A specific example of this sequence is shown in SEQ ID NO: 8.
There is no particular limitation as to the type of reporter gene to be used in this invention as long as its expression is detectable. Any reporter gene typically used in various assay systems by those skilled in the art can be employed, for example, the luciferase gene, the chloramphenicol acetyl transferase (CAT) gene, or the β-galactosidase gene.
Herein, "operably linked" means that the binding sequence and the reporter gene are linked such that binding of p53 to the p53-binding sequence triggers induction of reporter gene expression. Preferably, a minimal promoter sequence is arranged upstream of the reporter gene, and the p53-binding sequence is further upstream of this promoter sequence.
There is no particular limitation as to the type of cells which can be used for a screening of the present invention, and for example, cell lines available from American Type Culture Collection (ATCC) such as the SW480 colorectal adenocarcinoma cell line, H1299 non small lung carcinoma cell line, and MCF7 mammary carcinoma cell line can be used. A variety of methods known to those skilled in the art, for example, the use of electroporation and lipofectin reagent, can be used for the transduction of cells with a vector. The above-described reporter gene expression level can be measured by methods generally used by those skilled in the art and depending on the type of reporter gene used.
A compound that reduces or enhances reporter gene expression level when compared to that measured with the same method in the absence of the test sample, is selected as a candidate compound for an apoptosis-regulating agent. p53 is known to bind to a "p53-binding sequence", activate target gene transcription, and then induce apoptosis. Therefore, candidate compounds that reduce reporter gene expression level in the screening methods of the present invention are expected to be used as apoptosis inhibitors. Candidate compounds that enhance reporter gene expression level may be used as apoptosis accelerators.
Test samples to be screened include extracts of the above-mentioned cells, gene library expression products, synthetic low molecular weight compounds, synthetic peptides, and natural compounds.
When used medicinally, a protein or antibody of the present invention, or a compound isolated by a screening method of this invention, may be directly administered to patients, or administered in a dosage form prepared by drug-manufacturing methods well-known in the art. For example, the dosage form may be prepared by appropriately combining with a pharmaceutically acceptable carrier or medium such as sterilized water, physiological saline, a plant oil, an emulsifier, a suspending agent, a surfactant, or a stabilizer. Administration to patients may be carried out by methods known to those skilled in the art, for example, by intra-arterial, intravenous, or subcutaneous administration, or by intranasal, transbronchial, intramuscular, or oral administration. Although the dosage may vary depending on the route of administration, patient body weight and age, those skilled in the art can appropriately select a suitable dosage. In addition, when the compound is encoded by DNA, gene therapy can be performed using a gene therapy vector into which that DNA has been introduced. Those skilled in the art can select an appropriate dosage and method of administration depending on the patient body weight, age, symptoms, and such.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a photograph showing the result of Northern blot analysis in Example 1 of p53-induced transcripts along with a positive control (p21.sup.waf1) and an internal control (actin protein).
FIG. 2 shows (A) a comparison of predicted amino acid sequences encoded by the two cDNAs derived from alternative splicing of p53DINP1 chromosomal DNA (SEQ ID NOS: 2 and 4), and (B) the composition of the cDNAs compared with the genomic DNA.
FIG. 3 shows (A) the p53-binding sequence (SEQ ID NOS: 19 and 8, respectively) and its schematic representation, and (B) a photograph of electrophoresis showing the results of EMSA conducted in Example 3.
FIG. 4 is a graph showing the transcription inducing activity of the p53-binding sequence from the p53DINP1 gene using the luciferase gene as a reporter gene, as in Example 4.
FIG. 5 is a photograph showing confirmation of p53-dependent induction of p53DINP1.
FIG. 6 is a photograph showing confirmation of p53DINP1 induction by double-strand breaks caused by γ-ray irradiation or adriamycin treatment.
FIG. 7 represents photographs showing the nuclear localization of p53DINP1 protein expression.
FIG. 8 represents graphs and photographs showing (A) viable cell number, (B) distribution of cell cycle phase, and (C) expression of p53DINP1 and related proteins in cells, where each was measured after DNA damage such as double-strand breaks was inflicted in the presence of the p53DINP1 DNA antisense oligonucleotide (AS) or sense oligonucleotide (SE).
FIG. 9 represents graphs and photographs showing (A) p53AIP1 expression and p53 Ser46 residue phosphorylation, (B) cell cycle phase distribution, and (C) cells stained in TUNEL analysis, where each was measured after overexpression of both p53 and p53DINP1.
FIG. 10 represents a photograph showing recovery of a p53 Ser46 kinase by immunoprecipitation using the anti-p53DINP1 antibody.
BEST MODE FOR CARRYING OUT THE INVENTION
Identification of a Novel Transcript Induced by p53
The differential display method (Takei et al., 1998; Okamura et al., 1999, supra) was applied to identify a novel p53-induced transcript.
The LacSwitch vector system (Stratagene) (Wyborski and Short, 1991) was used as an expression system for p53, and the wild-type p53 gene was inserted into the vector thereof to construct a recombinant expression vector. In the same way, recombinant expression vectors containing a mutant type p53 gene or the chloramphenicol acetyltransferase (CAT) gene were constructed as controls. Each vector thus constructed was used for the transfection of SW480 cells deficient in wild-type p53. Each transfectant was exposed to 5 mM IPTG to induce gene expression, and mRNA was then extracted at zero, eight, 16, 24, 32, and 40 hours after this induction. mRNAs thus extracted and a variety of primer pairs were combined and used for differential display.
Of the several hundred bands detected by this method, a 250 bp DNA fragment was detected in the presence of wild-type p53 with a very strong signal, compared to the controls (data not shown). This fragment is hereinafter designated as 8T250.
This fragment was subcloned and its nucleotide sequence was determined using standard procedures. Comparison with known DNA nucleotide sequences in public databases did not reveal any DNA identical to the novel transcript designated as 8T250.
In order to confirm the result obtained by the above differential display, RT-PCR and Northern blot analysis were employed using the mRNAs obtained from the above-described SW480 cells, and those obtained from U373MG cells transformed with an adenovirus vector containing the wild-type p53 gene (Ad-p53) or LacZ gene (Ad-LacZ). Northern blot analysis was performed using a known method (el-Deiry, W. S., Nelkin, B. D., Celano, P., Yen, R. W., Falco, J. P., Hamilton, S. R., and Baylin, S. B. (1991). High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer. Proc. Natl. Acad. Sci. U.S.A. 88, 3470-3474). The primers used for RT-PCR were F1 (5'-TTGTGGGTGAAGTCAGTTCTT-3'/SEQ ID NO: 6) and R1 (5'-GAGCTTCCACTCTGGGACT-3'/SEQ ID NO: 7). In addition, the RT-PCR product obtained using the above primers was used as a probe for Northern blot analysis. FIG. 1 shows the result of Northern blot analysis of the mRNAs prepared using the U373MG cell line.
As shown in FIG. 1, "p53DINP1" transcript expression was specific to cells transfected with Ad-p53, and expression was observed immediately after Ad-p53 transfection. This result thus shows that p53DINP1 gene expression is p53-dependent, and that the transcript is induced immediately after induction by p53. In addition, the expression profile of the p53DINP1 transcript was the same as that of known molecule p21.sup.WAF1, whose expression is induced by p53.
Using Northern blot analysis, various tissues were then examined for the expression of the above transcript. The sixteen human tissues examined all expressed p53DINP1, and a relatively high level of expression was seen in the pancreas, spleen, testis, and peripheral leukocyte (data not shown). The size of the transcript was estimated to be about 6 kb throughout these experiments.
Isolation of the p53DINP1 Gene
In order to isolate a full length cDNA of the novel transcript detected in Example 1, a human thymus-derived cDNA library (1×106 colonies) was used to screen for the full length cDNA, using the 8T250 DNA fragment as a probe. The sixteen positive colonies obtained in this screening were subjected to nucleotide sequence determination, resulting in the detection of two open reading frames (ORFs). One ORF coded a polypeptide comprising 240 amino acids (SEQ ID NO: 2), and the other coded a polypeptide comprising 164 amino acids (SEQ ID NO: 4). Hereinafter, the gene generating the two ORFs is designated as p53DINP1 (p53-dependent Damage-Inducible Nuclear Protein 1). Hereinafter, the former and latter ORF are designated as genes p53DINP1a and p53DINP1b respectively.
In order to identify the p53DINP1 gene's chromosomal nucleotide sequence, a cosmid clone containing the complete p53DINP1 gene was isolated, and the nucleotide sequence was determined (SEQ ID NO: 5, accession NO: DDBJ/EMBL/GenBank AB062056). FIG. 2 (B) shows a comparison of the genomic DNA and the cDNA. The p53DINP1 gene comprises four exons. The two different transcripts (p53DINP1a and p53DINP1b) are generated by alternative splicing. FIG. 2 (A) shows a comparison of the amino acid sequences of p53DINP1a (SEQ ID NO: 1 and 2, accession NO: DDBJ/EMBL/GenBank AB017926) and p53DINP1b (SEQ ID NO: 3 and 4, accession NO: DDBJ/EMBL/GenBank AB017927).
More over, FISH analysis revealed that the genomic sequence spans a 15 kb genomic region within the chromosome 8q22 band (data not shown).
p53-Binding Site of the p53DINP1 Gene
The inventors found an essential p53-binding site (p53BS) comprising 20 nucleotides (SEQ ID NO: 8) in intron 2 of the p53DINP1 gene. This binding site was 85% identical to the consensus p53-binding sequence (FIG. 3). In order to examine whether p53 binds to oligonucleotides corresponding to the p53BS sequence, electrophoretic-mobility shift assays (EMSA) were employed as described below.
H1299 lung carcinoma cells were infected with a recombinant adenovirus vector that expresses wild-type p53 (Ad-p53), and a nucleic acid extract was then prepared from these infected cells. To this nucleic acid extract was added sonicated salmon sperm DNA (0.5 μg), EMSA buffer, and an appropriate 32P-labeled double-stranded oligomer (pre-annealed). This mixture was then incubated at room temperature for 30 minutes. The mixture was incubated in the presence of anti-p53 monoclonal antibodies, Pab421 (Oncogene Science) and/or Pab1801 (Santa Cruz Biotechnology). An unlabeled oligonucleotide corresponding to the p53BS sequence (designated as "self" in FIG. 3) or an unlabeled non-specific oligonucleotide (designated as "TL" in FIG. 3) was also added to some samples as a competitor. After incubation, each sample was subjected to electrophoresis on a 4% polyacrylamide gel containing 0.5×TBE. Following electrophoresis the gel was dried and subjected to autoradiography at -80° C. for three hours.
FIG. 3 shows the result of EMSA. Mobility of the 20 bp oligonucleotide shifted when it was mixed with the nucleic acid extract (FIG. 3). An additional mobility shift was observed when anti-p53 monoclonal antibodies p53Ab1 (Pab421) and p53Ab2 (Pab1801) were added to the mixture, indicating that the p53DINP1 gene's 20 bp oligonucleotide sequence is the p53-binding site.
p53DINP1 as a Target Gene for p53
To examine whether the p53-binding sequence identified in Example 3 has p53-dependent transcription-enhancing activity, a reporter assay using the luciferase gene was employed as described below.
A 477 bp fragment containing the wild-type p53-binding sequence (GAACTTGGGGGAACATGTTT: SEQ ID NO: 8) within intron 2 was amplified by PCR using primer F (CGCCGAGCTCCCTGCAATACTCACACTGC: SEQ ID NO: 9) and primer R (CAGTACGCGTCCTCCATAAGACCCCAATA: SEQ ID NO: 10). The amplified PCR products were then cloned upstream of the SV40 minimal promoter sequence within the pGL3-promoter vector (Promega, Madison, Wis., USA). Hereinafter, the recombinant reporter vector thus obtained is designated as "intron 2-wt".
An oligomer pair corresponding to one copy of the p53-binding sequence, 1F (CGAACTTGGGGGAACATGTTTA: SEQ ID NO: 11) and 1R (CGCGTAAACATGTTCCCCCAAGTTCGAGCT: SEQ ID NO: 12); and another oligomer pair corresponding to two copies of the p53-binding sequence, 2F (CGAACTTGGGGGAACATGTTTGAACTTGGGGGAACATGTTTA: SEQ ID NO: 13) and 2R (CGCGTAAACATGTTCCCCCAAGTTCAAACATGTTCCCCCAAGTTCGAGCT: SEQ ID NO: 14); were separately and respectively annealed. The annealed oligomer pairs were then ligated with pGL3-promoter vectors that had been cleaved with MluI and XhoI, yielding recombinant reporter vectors (designated as "X1" and "X2" respectively).
A vector comprising a mutation within p53BS ("intron 2-mt") was constructed by using a QuickChange Site-Directed Mutagenesis Kit (Stratagene) to introduce a point mutation where p53BS's fourth "C" nucleotide was substituted with "T". The mutant form of the intron 2 fragment (477 bp) was cloned into the pGL3-promoter vector in the same way as for the above-mentioned wild-type.
Each recombinant reporter vector thus constructed was used for cotransfection of H1299 lung carcinoma cells (p53-/-) with a plasmid expressing wild-type or mutant type p53. After transfection with a vector or the like, quantitative and relative final luciferase activities in the cells were measured using a luminometer and according to the attached protocol (Promega).
FIG. 4 shows the results of these measurements. Relative luciferase activity increased significantly when cells were cotransfected with the intron 2-wt reporter vector and the wild-type p53 expression vector. However, this kind of increased activity was not observed when the mutant type p53 or intron 2-mt was introduced.
Luciferase activity increased several times when the X1 reporter vector was introduced with the wild-type p53 expression vector. When the X2 reporter vector was used with wild type p53, luciferase activity increased more than 30 times.
The results from these luciferase assays and from EMSA in Example 3 clearly indicate that p53DINP1 is a direct p53 target.
Genotoxic Stress- and p53-Dependent Induction of p53DINP1
In order to examine whether endogenous p53 induces p53DINP1, mouse p53+/+MEF cells or p53-/- MEF cells were irradiated with γ-rays and expression of the mouse p53DINP1 gene in response to DNA damage was analyzed. FIG. 5 shows these results.
As shown in FIG. 5, there was no sign of p53DINP1 mRNA expression in p53-/-MEF cells following DNA damage. On the other hand, there was a significant increase of p53DINP1 mRNA expression in p53+/+MEF cells in response to DNA damage by γ-ray. These results indicate that DNA damage-induced p53DINP1 expression is dependent on endogenous p53.
An anti-p53DINP1 polyclonal antibody was prepared for use in Western blotting to examine the expression of endogenous p53DINP1 in a human cell line in which a variety of DNA damage had been inflicted.
The above-mentioned anti-p53DINP1 polyclonal antibody was prepared by producing p53DINP1 protein in a recombinant bacterium, using this protein to immunize rabbits using standard methods, harvesting serum from the immunized rabbits, and then affinity-purifying the serum using the above-mentioned p53DINP1 protein.
DNA-damage of the human cell line was carried out as follows: MCF7 cells (mammary carcinoma) and H1299 cells were either treated with 3 μM adriamycin for two hours, or with γ-rays (14 Gy) or UV radiation (10 J/m2). Following this process, each cell-line was cultured in the absence of the damaging agent, and harvested at appropriate intervals. Western blotting was used to measure the expression of p53DINP1 in the damaged cells thus harvested.
For Western blotting, harvested cells were dissolved in a lysis buffer, and a portion (50 μl) of the cell lysate containing soluble cellular proteins was subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (HybondTMECL®). The membrane carrying the separated proteins was treated with a blocking solution containing 5% defatted milk powder (Carnation, Nestle) in TBS-T buffer at room temperature for two hours, and then incubated with anti-p53DINP1 antibody or anti-p53 antibody at room temperature for one hour. After washing, the membrane was incubated with a secondary antibody (peroxidase-bound sheep anti-rabbit Ig antibody), and finally the desired proteins including p53DINP1 were detected using ECL (Amersham Pharmacia Biotech).
As shown in FIG. 6, p53DINP1 expression was significantly induced within four hours of DNA damage by factors triggering double-strand breaks (γ-ray irradiation and adriamycin). p53DINP1 expression was also induced after DNA damage by UV irradiation, however in comparison with other inducers, expression was delayed and relatively moderate.
p53 expression was measured at the same time using the anti-p53 antibody, and was rapid and significant in response to all treatments, regardless of the type of DNA-damaging agent. The difference between expression induction of p53 and p53DINP1 in response to DNA-damaging treatment suggests that induction of the p53DINP1 protein mediates at least two different p53 activation mechanisms.
p53DINP1 as a Nuclear Protein
Immunocytometry was carried out to analyze the intracellular localization of p53DINP1 protein in MCF7 cells.
MCF7 cells were placed on a poly-D-lysine-coated multi-well chamber slide (Becton Dickinson), and fixed with 4% paraformaldehyde in PBS. The cells were rendered permeable by treatment with 0.2% NP-400 in PBS at room temperature for eight minutes, then blocked in a blocking solution (2.55% horse serum albumin in PBS) at room temperature for 30 minutes. The cells were then incubated with TBS-T containing 1 μg/ml anti-p53DINP1 antibody. This antibody was stained with Congo red-conjugated goat anti-rabbit secondary antibody, followed by DAPI staining. After staining, the cells were viewed with an ECLIPSE E600 microscope (Nikon). As depicted in FIG. 7, cell nuclei staining revealed a clear focus pattern, indicating that p53DINP1 expression is localized to nuclei.
Involvement of p53DINP1 in Apoptosis Induced by Double-Strand Breaks
In order to clarify the role of p53DINP1 in cell death after DNA damage by double-strand breaks, antisense oligonucleotides (AS1 and AS2) were used in cell damage experiments as follows.
Antisense oligonucleotides AS1 (TGGAACATTGTTAAGG: SEQ ID NO: 15) and AS2 (TCAGCCTCTGGAACAT: SEQ ID NO: 16) were prepared in accordance with the p53DINP1 gene nucleotide sequence and in order to inhibit endogenous p53DINP1 expression. Sense oligonucleotides SE1 (CCTTAACAATGTTCCA: SEQ ID NO: 17) and SE2 (ATGTGCCAGAGGCTGA: SEQ ID NO: 18) were also prepared as controls.
MCF7 cells (2×106) were plated onto a 10 cmφ dish and the next day the above-prepared antisense or sense oligonucleotides (1 μM) were introduced into cells using Lipofectin reagent (GibcoBRL). The cells were incubated at 37° C. for four hours, the culture medium was replaced, and the cells were then damaged using either γ-irradiation (30 Gy) or exposure to 3 μM adriamycin. After this genotoxic stress, damaged cells were harvested at appropriate intervals and RNA expression and intracellular proteins were analyzed using RT-PCR and Western blotting respectively.
FIG. 8 (C) shows the result of Western blotting. From twelve hours after DNA damage (double strand breaks (DSB) caused by γ-irradiation or DNA damage caused by UV-irradiation), the sense oligonucleotide did not affect p53DINP1 protein expression in MCF7 cells. On the other hand, the antisense oligonucleotide (AS2) clearly suppressed this expression.
AS2 and SE2 were then used to examine whether this suppression of p53DINP expression by the antisense oligonucleotide would affect cell death after double-strand breaks. This is outlined below:
AS2 or SE2 were introduced into exponentially growing MCF7 cells and incubated for four hours. These cells were then irradiated with γ-rays (30 Gy) and harvested immediately using trypsin treatment. The damaged cells were diluted to 1×106 cells per dish and maintained in a 6 cmφ dish with fresh medium. The cells were harvested 48 and 72 hours after DNA damage, and cell viability was assessed by staining with 0.4% tripan blue.
As shown in FIG. 8 (A), the viability of cells treated with SE2 decreased to 40% or less 48 hours after DNA damage, however increased to 50% or more for cells treated with AS2. Thus, p53DINP1 antisense oligonucleotides can suppress cell death caused by DNA damage, suggesting that p53DINP1 plays a role in apoptosis induction.
Cell viability was then measured using cell cycle analysis. MCF7 cells were damaged as described above, trypsinized 72 hours later, and then washed and fixed with 70% ethanol. The fixed samples were centrifuged, treated with 1 mg/ml RNase and resuspended in 50 μg/ml propidium iodide. The stained cells were then analysed on a FACScan flow cytometer (Beckton-Dickinson).
As FIG. 8 (B) shows, 33% of cells treated with SE were dead 72 hours after γ-irradiation (sub-G1), however this was suppressed to 16.5% of cells treated with AS. Similarly, 72 hours after adriamycin treatment, cell death in cells treated with SE was 21.3% or more, but only 11.3% in cells treated with AS. AS pretreatment had no effect on induction of apoptotic cell death caused by UV irradiation (data not shown). These results suggest that p53DINP1 is involved in a p53-dependent apoptosis signal caused by double-strand breaks.
Thus if p53DINP1 is involved in p53-dependent apoptosis, then inhibition of p53DINP1 expression might affect the functions of p53AIP1. Accordingly, the inventors examined whether p53AIP1 expression is induced in AS- or SE-treated MCF7 cells after DNA damage caused by γ-irradiation or adriamycin treatment.
At the same time, expression levels of other p53 target genes and apoptosis-related genes were measured in MCF7 cells using the same methods as described above. FIG. 8 (C) shows the results. Of the genes tested, only p53AIP1 expression was suppressed by inhibition of p53DINP1 expression. There was no sign of change in expression of p21.sup.waf1, BAX (FIG. 8 (C)), caspase-3, caspase-9, and MDM2 (data not shown for these last three).
Thus, since inhibition of p53DINP1 expression by antisense oligonucleotide treatment suppresses cell death in response to double-strand breaks, and also suppresses p53AIP1 expression, it was suggested that p53DINP1 regulates p53AIP1 expression through p53 phosphorylation at the Ser46 residue. Accordingly, the inventors examined whether the inhibition of p53DINP1 expression by treatment with antisense oligonucleotides affected the phosphorylation of p53 protein.
MCF7 cells pretreated with AS or SE were damaged with γ-irradiation, harvested at the above-described times, and then subjected to Western blotting analysis using a phosphorylated residue-specific antibody against Ser15, Ser20, or Ser46.
As shown in FIG. 8 (C), Ser15 and Ser20 phosphorylation levels (data not shown for Ser20) decreased in parallel with the amount of p53 protein, and inhibition of p53DINP1 expression did not affect phosphorylation. However, Ser46 phosphorylation was almost completely suppressed by the inhibition of p53DINP1 expression caused by AS2 treatment. This result suggests that p53DINP1 activates the p53 protein such that apoptosis is induced by regulation of p53 Ser46 phosphorylation. This result also suggests that such a modification of the p53 protein activates transcription of other apoptosis-inducing genes such as p53AIP1.
In contrast to γ-irradiation, UV irradiation of MCF7 cells pretreated with AS or SE as described above did not suppress p53AIP1 expression, but significantly induced p53 phosphorylation at Ser46, as shown in FIG. 8 (C). These results suggest that at least two pathways are involved in p53 Ser46 phosphorylation and p53AIP1 induction; one involved in double-strand breaks, and the other concerning DNA damage caused by UV irradiation and such. p53DINP1 may be involved in the former of these.
Enhancement of p53 Ser46 Phosphorylation, p53AIP1 Expression, and Apoptotic Cell Death, by Overexpression of p53 and p53DINP1
To examine the ability of p53DINP1 to induce apoptosis as a cofactor for p53 Ser46 phosphorylation, the following experiments were conducted. MCF7 cells (2×106) harboring wild-type p53 protein were plated on a 10 cmφ dish. The next day, the cells were transfected either with a combination of wild-type p53 expression vector and p53DINP1 expression vector (pcDNA3.1(+)/p53DINP1); or a combination of wild-type p53 expression vector and an empty vector for p53DINP1 (pcDNA3.1(+)), using FuGene® 6 transfection reagent (Roche) according to the attached protocol. The cells were incubated at 37° C. for four hours. To activate p53, the DNA of the cells was then damaged using γ-irradiation (30 Gy) or exposure to 3 μM adriamycin. Following administration of this genotoxic stress, damaged cells were harvested at a variety of times and analyzed using RT-PCR, Western blotting, cell cycle analysis, and TUNEL assays. TUNEL (terminal transferase-mediated dUTP nick end-labelling) assays were performed in situ using Apoptag Direct (Oncor) according to the attached protocol. Finally, cells were stained with propidium iodide and immunofluorescence was viewed using an ECLIPSE E600 microscope (Nikon).
As FIG. 9 (A) illustrates, Western blotting analysis revealed that overexpression of both p53DINP1 and p53 stimulated induction of p53 Ser46 phosphorylation and p53AIP1 expression. FIGS. 9 (B and C) shows the results of apoptotic cell number evaluation. TUNEL analysis (FIG. 9 (C)) and FACS analysis (FIG. 9 (B)) revealed that overexpression of both p53DINP1 and p53 significantly increased the proportion of apoptotic cells by 72 hours after the administration of genotoxic stress to induce double-strand breaks.
Interaction of p53DINP1 with p53-Ser46 Kinase
In order to clarify the governing mechanism of p53DINP1-mediated Ser46-phosphorylation of p53, in vitro kinase analysis was performed using immunoprecipitated p53DINP1 and GST-p53.
p53DINP1 and its related proteins were immunoprecipitated from the cell lysate of MCF7 cells, which were either treated or untreated with radiation, using a rabbit anti-p53DINP1 polyclonal antibody. The phosphorylation activity of the resultant precipitates was then analyzed. The p53DINP1-specific antibody used in this experiment had previously been used to effectively precipitate p53DINP1 from crude cell lysates (data not shown).
As shown in FIG. 10, Ser46 phosphorylation was clearly accelerated in immunoprecipitates prepared from cell lysates isolated four, eight, and twelve hours after cell damage. In contrast, Ser46 kinase activity was not observed in the control, which comprised using rabbit IgG prepared prior to immunization to immunoprecipitate proteins from cells isolated twelve hours after cell damage ("control IgG" in FIG. 10).
p53DINP1's specificity to Ser46 phosphorylation was evaluated by analyzing p53 phosphorylated at Ser20 and Ser33 using the rabbit polyclonal antibodies p53-P-Ser20 (which recognizes Ser20-phosphorylation of p53) and p53-P-Ser33 (which recognizes Ser33-phosphorylation of p53).
Ser33 is reported to be another possible phosphorylation site which is important in p53-dependent apoptosis (Bulavin, D. V., Saito, S., Hollander, M. C., Sakaguchi, K., Anderson, C. W., Appella, E., and Formace, A. J. Jr. (1999). Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV irradiation. EMBO J. 18, 6845-6854). However, as FIG. 9 indicates, phosphorylation of Ser20 and Ser33 was not observed in analysis of whole cell lysates derived from irradiated MCF7 cells. The result suggests that p53DINP1 is involved in the specific phosphorylation of Ser46. Results from the immunoprecipitation experiment using the rabbit anti-p53DINP1 antibody also suggest that the kinase which phosphorylates p53 at Ser46 also interacts with p53DINP1 in response to DNA damage.
The present invention provides "p53DINP1", a novel target gene for the p53 tumor suppressor protein. This gene is closely associated with p53-mediated apoptosis caused by DNA damage such as double-strand breaks. This gene is particularly involved in phosphorylation of p53 at Ser46, an upstream signal in the apoptosis pathway. Therefore, the p53DINP1 gene and p53DINP1 protein of the present invention would be useful as a tool for research into the induction of p53-dependent apoptosis, and also for apoptosis-mediated therapy for human cancer cells by introducing the gene of the present invention into cancer cells using an expression vector or an adenovirus vector. The p53DINP1 gene of the present invention, and the protein encoded by this gene, can also be used as target molecules in the development of therapeutic agents for apoptosis-associated diseases.
The present invention also provides a method of screening for candidate compounds that can control apoptosis induction. Thus, the development of preventive and therapeutic agents that comprise such a compound as their effective component, for both cancers and apoptosis-associated diseases, are greatly expected.
1915313DNAHomo sapiensCDS(40)..(762)/codon_start=1 /gene="p53DINP1" /product="p53DINP1a" 1taaaagacat ccagccaaac tctcagtctt gccttaaca atg ttc cag agg ctg 54 Met Phe Gln Arg Leu 1 5aat aaa atg ttt gtg ggt gaa gtc agt tct tcc tcc aac caa gaa cca 102Asn Lys Met Phe Val Gly Glu Val Ser Ser Ser Ser Asn Gln Glu Pro 10 15 20gaa ttc aat gag aaa gaa gat gat gaa tgg att ctt gtt gac ttc ata 150Glu Phe Asn Glu Lys Glu Asp Asp Glu Trp Ile Leu Val Asp Phe Ile 25 30 35gat act tgc act ggt ttc tca gca gaa gaa gaa gaa gaa gag gag gac 198Asp Thr Cys Thr Gly Phe Ser Ala Glu Glu Glu Glu Glu Glu Glu Asp 40 45 50atc agt gaa gag tca cct act gag cac cct tca gtc ttt tcc tgt tta 246Ile Ser Glu Glu Ser Pro Thr Glu His Pro Ser Val Phe Ser Cys Leu 55 60 65ccg gca tct ctt gag tgc ttg gct gat aca agt gat tcc tgc ttt ctc 294Pro Ala Ser Leu Glu Cys Leu Ala Asp Thr Ser Asp Ser Cys Phe Leu 70 75 80 85cag ttt gag tca tgt cca atg gag gag agc tgg ttt atc acc cca ccc 342Gln Phe Glu Ser Cys Pro Met Glu Glu Ser Trp Phe Ile Thr Pro Pro 90 95 100cca tgt ttt act gca ggt gga tta acc act atc aag gtg gaa aca agt 390Pro Cys Phe Thr Ala Gly Gly Leu Thr Thr Ile Lys Val Glu Thr Ser 105 110 115cct atg gaa aac ctt ctc att gaa cat ccc agc atg tct gtc tat gct 438Pro Met Glu Asn Leu Leu Ile Glu His Pro Ser Met Ser Val Tyr Ala 120 125 130gtg cat aac tcc tgc cct ggt ctc agt gag gcc acc cgt ggg act gat 486Val His Asn Ser Cys Pro Gly Leu Ser Glu Ala Thr Arg Gly Thr Asp 135 140 145gaa tta cat agc cca agt agt ccc aga gtg gaa gct caa aat gaa atg 534Glu Leu His Ser Pro Ser Ser Pro Arg Val Glu Ala Gln Asn Glu Met150 155 160 165ggg cag cat att cat tgt tat gtt gca gct ctt gct gct cat aca act 582Gly Gln His Ile His Cys Tyr Val Ala Ala Leu Ala Ala His Thr Thr 170 175 180ttt ctg gaa caa ccc aag agc ttt cgc cct tcc cag tgg ata aaa gaa 630Phe Leu Glu Gln Pro Lys Ser Phe Arg Pro Ser Gln Trp Ile Lys Glu 185 190 195cac agt gaa aga cag cct ctt aac aga aat agc ctt cgt cgc caa aat 678His Ser Glu Arg Gln Pro Leu Asn Arg Asn Ser Leu Arg Arg Gln Asn 200 205 210ctt acc agg gat tgc cac cct cgg caa gtc aag cac aat ggc tgg gtt 726Leu Thr Arg Asp Cys His Pro Arg Gln Val Lys His Asn Gly Trp Val 215 220 225gtt cat cag ccc tgc ccg cgt cag tac aat tac taa tagtttcaag 772Val His Gln Pro Cys Pro Arg Gln Tyr Asn Tyr230 235 240ttttgttggt tggtttctct tggtttgtgc taacatgtat ggatgtgtgt atatgtacag 832tgaaaatgtt gtctctttac aaccaattga taaccaatca catagtttta tcagtgtatt 892tagacactat cttgaaaatc agatttatat gctgtgtatc acataatgcc ttgcctttaa 952catttacttt ttttgtacac tttttcagat tatttctgga aacatatcaa tataattaca 1012gtgtttgggg gtgtctttaa atatattagg ttatacatta gtcagcattt taaagacatt 1072tcttcccaag tacgagaata ggcatctttc attttcattt tattttgtat tacttaatct 1132tttaagcaag caaaaattta ttctcagggt cagctgtaca ctttattgac cagtacttga 1192taatctctct gtatatgatg aatacatttt tatacactaa cattagcatt aacaggtgat 1252agttgccatg gatataatgg aattatggct ggactttctt ttgaaagaaa acttgatgta 1312ttctgtgtgt atggtttttc cccagattag tcatacagtt catttggaat tcaggtacat 1372taagctttag tgaagagtgc atgcagtaat tccaatgtga ctgcatgacg tggtacagac 1432attacaggtg ttgtagacag aggcacttgt ctcgtgcaga gggattaaat tagacctgtg 1492agattatatt tggaaaaatt catgtctgta actaacccat tagtgcagta tttaatttgt 1552tactattcct tcccgccaat tctgtccact cctcacctcg catcagctat aaatttggaa 1612atacttgtcc aggcactcaa gtgacttcat atttctctct gcccatggga aaagagatag 1672gctttatatt tccacagant gaaaaatcct ctgtcatgga gcctgtcctg ccaagtggca 1732agaatgtggg gactgtctgg tgatgatgtc tttcatggca tctgagtgaa gaatgacagg 1792ttggctcaac ttttttcttt ttttttttta attgccttgt attgtaagta ttcttccctg 1852cagtccaagt gacttttcat tttttgtttt aacttcaggc aaaatcttta accactctgg 1912cctctgtttc ccccaccaac ggggagcagt gacatttacc tccctcacag agtcactgtg 1972aggattctat actgatttga agtggagctg ttcagaactg aaccttgtag gaaattccaa 2032gggcctttct actgaatctg gtgatggggt ggggccgtgg cactttctct gccacagctg 2092ttcttcacag tgttggtgct aatgaggcca gggtgcaggg ttcgattcac acgtaggcca 2152gttaacttag agaaaatcta tttccttacc tctagccagt cacttccttt ttccgcagtt 2212gtgatgggtt ttgctgagcc atccactctg actgatttcc tctgaagtaa acatatttac 2272aatccaaagc aattctactg acagaagtgt tgccttcata atcaaacagc ttgtttttcc 2332atctcctctg caaccctaat taaatgagta caggtctaca aaatgttttc aaggagaaaa 2392gcagcatatc cttaagtgaa gtattatatt tttcaataac cctgtagtgg cttgatgcag 2452ggaaccctgg gggactttca gcgaagagct gtgctctttt ctgactagat tagagcgttt 2512ggagtggaag acgtcaaatg tgtagtgaga tggaggtttt acattgttct tctactggct 2572gtgatgaagt gccagaatgt ctctttagaa caagagttag attccccctt tctccttatt 2632gccccttccg ttttgacttc ccctttattt atttgctgtc taattagggg ccaagtctgt 2692aaagttttgt caaagtgagt tagaagttgt tttttcttac tatttgtgtt taccagagtt 2752gggagataag atagtttcca tgaaggtgtg tatgttttat acgatgtttg ttatagggcc 2812atgcattggt aacttgaaaa taagacagct taatgtcttc aggatgtaat cagacagctt 2872aatgtcttca gggatgtaaa actctgacta cacggcgtct ctttttcata cattgcatgt 2932aagttgttag tacctcacaa gctacagaag ttcagccatg agattttgtt tggcaacatg 2992aacagatttg tgtataactg caatggcctt ttttccagat ttccttattg actttttgtt 3052tgccttacct ggggctagtt ttttatgctt tgtacctaga aaacaaaaaa ttacattcgt 3112tgggcttttt ttcaaggttg ggattaccac accacctgga atatcatact gtggtttctg 3172cctaaaattg gcacatgtaa gtattgaaga aaatggttat ataattcagt tgaaactctt 3232ggttattaga tgttaggcat ctcctgtatg taagacacaa ggccaaccac aacacagaac 3292gatgttgacc tgttaagtat tctctgaaac atggccaaaa tgcattttat gagctttttt 3352tttttgctat tgtaaatatt agtggtttac aatgcgcttt aaacatattt ctttaaaatg 3412caagcagtga gaaataagac ctctctgaat tagtagctct aaactgttaa catagaatgt 3472tacttggaaa aagtctggaa tatgtggtgt acacaagcag tgcttcgtga atgagtttct 3532tagcttttat agtgcgccat gtttctcaaa gtttgttttt gttgacaaaa cattttataa 3592tatatatctt atgtttattt tttttctcaa ctaattgtgt actgcactgt aaggtgaaaa 3652ttagccatcc attatttatc ttctgtggca atgcatttat atggttgatt gggtggggaa 3712ttttttgcag aaagatgcaa agtgatgggt tttcgacttc ctatcgcagg gagcttttaa 3772gaaatattaa tttcctatac atttttccaa tccccatgca aactgttcct gtttacatac 3832cttctctgtt gtatcagtac tttgagtgag aagacagttt atttaaaact tgagcaggct 3892gttcagcatt ttttctgctt ctgaaatctg tatagtacac tggtttgtaa tcattatgtc 3952ttcattgaaa tccttgctac ttctcttcct cctcaatgaa atacattata tattatcttt 4012atgtactctt aagaaaaacg agcaaggaag agtatcttca ttattctcat tttctctgag 4072ttggaaacaa aaacatgaag gactccaact agaagacaga tatttacatt taaatagatt 4132agtgggaaaa ctttaagagt ttccacatat tagttttcat tttttgagtc aagagactgc 4192tccttgtact gggagacact agtagtatat gtttgtaatg ttactttaaa attatctttt 4252tattttataa ggcccataaa tactggttaa actctgttaa aagtgggcct tctatcttgg 4312atggtttcac tgccatcagc catgctgata tattagaaat ggcatcccta tctacttact 4372ttaatgctta aaattataca taaaatgctt tatttagaaa acctacatga tacagtggtg 4432tcagccttgc catgtatcag tttcacttga aatttgagac caattaaatt tcaactgttt 4492agggtggaga aagaggtact ggaaaacatg cagatgagga tatcttttat gtgcaacagt 4552atcctttgca tgggaggaga gttactcttg aaaggcaggc agcttaagtg gacaatgttt 4612tgtatatagt tgagaatttt acgacacttt taaaaattgt gtaattgtta aatgtccagt 4672tttgctctgt tttgcctgaa gttttagtat ttgttttcta ggtggacctc tgaaaaccaa 4732accagtacct ggggaggtta gatgtgtgtt tcaggcttgg agtgtatgag tggttttgct 4792tgtattttcc tccagagatt ttgaacttta ataattgcgt gtgtgttttt ttttttttta 4852agtggctttg tttttttttc tcaagtaaaa ttgtgaacat atttccttta taggggcagg 4912gcatgagtta gggagactga agagtattgt agactgtaca tgtgccttct taatgtgttt 4972ctcgacacat tttttttcag taacttgaaa attcaaaagg gacatttggt taggttactg 5032tacatcaatc tatgcataaa tggcagcttg ttttcttgag ccacggtcta aattttgttt 5092ttatagaaat tttttatact gattggttca tagatggtca gttttgtaca cagactgaac 5152aatacagcac tttgccaaaa atgagtgtag cattgtttaa acattgtgtg ttaacacctg 5212ttctttgtaa ttgggttgtg gtgcattttg cactacctgg agttacagtt ttcaatctgt 5272cagtaaataa agtgtccttt aacttcaaaa aaaaaaaaaa a 53132240PRTHomo sapiens 2Met Phe Gln Arg Leu Asn Lys Met Phe Val Gly Glu Val Ser Ser Ser 1 5 10 15Ser Asn Gln Glu Pro Glu Phe Asn Glu Lys Glu Asp Asp Glu Trp Ile 20 25 30Leu Val Asp Phe Ile Asp Thr Cys Thr Gly Phe Ser Ala Glu Glu Glu 35 40 45Glu Glu Glu Glu Asp Ile Ser Glu Glu Ser Pro Thr Glu His Pro Ser 50 55 60Val Phe Ser Cys Leu Pro Ala Ser Leu Glu Cys Leu Ala Asp Thr Ser 65 70 75 80Asp Ser Cys Phe Leu Gln Phe Glu Ser Cys Pro Met Glu Glu Ser Trp 85 90 95Phe Ile Thr Pro Pro Pro Cys Phe Thr Ala Gly Gly Leu Thr Thr Ile 100 105 110Lys Val Glu Thr Ser Pro Met Glu Asn Leu Leu Ile Glu His Pro Ser 115 120 125Met Ser Val Tyr Ala Val His Asn Ser Cys Pro Gly Leu Ser Glu Ala 130 135 140Thr Arg Gly Thr Asp Glu Leu His Ser Pro Ser Ser Pro Arg Val Glu145 150 155 160Ala Gln Asn Glu Met Gly Gln His Ile His Cys Tyr Val Ala Ala Leu 165 170 175Ala Ala His Thr Thr Phe Leu Glu Gln Pro Lys Ser Phe Arg Pro Ser 180 185 190Gln Trp Ile Lys Glu His Ser Glu Arg Gln Pro Leu Asn Arg Asn Ser 195 200 205Leu Arg Arg Gln Asn Leu Thr Arg Asp Cys His Pro Arg Gln Val Lys 210 215 220His Asn Gly Trp Val Val His Gln Pro Cys Pro Arg Gln Tyr Asn Tyr225 230 235 24035351DNAHomo sapiensCDS(40)..(534)/codon_start=1 /gene="p53DINP1" /product="p53DINP1b" 3taaaagacat ccagccaaac tctcagtctt gccttaaca atg ttc cag agg ctg 54 Met Phe Gln Arg Leu 1 5aat aaa atg ttt gtg ggt gaa gtc agt tct tcc tcc aac caa gaa cca 102Asn Lys Met Phe Val Gly Glu Val Ser Ser Ser Ser Asn Gln Glu Pro 10 15 20gaa ttc aat gag aaa gaa gat gat gaa tgg att ctt gtt gac ttc ata 150Glu Phe Asn Glu Lys Glu Asp Asp Glu Trp Ile Leu Val Asp Phe Ile 25 30 35gat act tgc act ggt ttc tca gca gaa gaa gaa gaa gaa gag gag gac 198Asp Thr Cys Thr Gly Phe Ser Ala Glu Glu Glu Glu Glu Glu Glu Asp 40 45 50atc agt gaa gag tca cct act gag cac cct tca gtc ttt tcc tgt tta 246Ile Ser Glu Glu Ser Pro Thr Glu His Pro Ser Val Phe Ser Cys Leu 55 60 65ccg gca tct ctt gag tgc ttg gct gat aca agt gat tcc tgc ttt ctc 294Pro Ala Ser Leu Glu Cys Leu Ala Asp Thr Ser Asp Ser Cys Phe Leu 70 75 80 85cag ttt gag tca tgt cca atg gag gag agc tgg ttt atc acc cca ccc 342Gln Phe Glu Ser Cys Pro Met Glu Glu Ser Trp Phe Ile Thr Pro Pro 90 95 100cca tgt ttt act gca ggt gga tta acc act atc aag gtg gaa aca agt 390Pro Cys Phe Thr Ala Gly Gly Leu Thr Thr Ile Lys Val Glu Thr Ser 105 110 115cct atg gaa aac ctt ctc att gaa cat ccc agc atg tct gtc tat gct 438Pro Met Glu Asn Leu Leu Ile Glu His Pro Ser Met Ser Val Tyr Ala 120 125 130gtg cat aac tcc tgc cct ggt ctc agt gag gcc acc cgt ggg act gat 486Val His Asn Ser Cys Pro Gly Leu Ser Glu Ala Thr Arg Gly Thr Asp 135 140 145gaa tta cat agc cca agt agt ccc agg gcc agg aaa agc tgc tta taa 534Glu Leu His Ser Pro Ser Ser Pro Arg Ala Arg Lys Ser Cys Leu150 155 160 165nactcacggg cacagaagtg gaagctcaaa atgaaatggg gcagcatatt cattgttatg 594ttgcagctct tgctgctcat acaacttttc tggaacaacc caagagcttt cgcccttccc 654agtggataaa agaacacagt gaaagacagc ctcttaacag aaatagcctt cgtcgccaaa 714atcttaccag ggattgccac cctcggcaag tcaagcacaa tggctgggtt gttcatcagc 774cctgcccgcg tcagtacaat tactaatagt ttcaagtttt gttggttggt ttctcttggt 834ttgtgctaac atgtatggat gtgtgtatat gtacagtgaa aatgttgtct ctttacaacc 894aattgataac caatcacata gttttatcag tgtatttaga cactatcttg aaaatcagat 954ttatatgctg tgtatcacat aatgccttgc ctttaacatt tacttttttt gtacactttt 1014tcagattatt tctggaaaca tatcaatata attacagtgt ttgggggtgt ctttaaatat 1074attaggttat acattagtca gcattttaaa gacatttctt cccaagtacg agaataggca 1134tctttcattt tcattttatt ttgtattact taatctttta agcaagcaaa aatttattct 1194cagggtcagc tgtacacttt attgaccagt acttgataat ctctctgtat atgatgaata 1254catttttata cactaacatt agcattaaca ggtgatagtt gccatggata taatggaatt 1314atggctggac tttcttttga aagaaaactt gatgtattct gtgtgtatgg tttttcccca 1374gattagtcat acagttcatt tggaattcag gtacattaag ctttagtgaa gagtgcatgc 1434agtaattcca atgtgactgc atgacgtggt acagacatta caggtgttgt agacagaggc 1494acttgtctcg tgcagaggga ttaaattaga cctgtgagat tatatttgga aaaattcatg 1554tctgtaacta acccattagt gcagtattta atttgttact attccttccc gccaattctg 1614tccactcctc acctcgcatc agctataaat ttggaaatac ttgtccaggc actcaagtga 1674cttcatattt ctctctgccc atgggaaaag agataggctt tatatttcca cagantgaaa 1734aatcctctgt catggagcct gtcctgccaa gtggcaagaa tgtggggact gtctggtgat 1794gatgtctttc atggcatctg agtgaagaat gacaggttgg ctcaactttt ttcttttttt 1854tttttaattg ccttgtattg taagtattct tccctgcagt ccaagtgact tttcattttt 1914tgttttaact tcaggcaaaa tctttaacca ctctggcctc tgtttccccc accaacgggg 1974agcagtgaca tttacctccc tcacagagtc actgtgagga ttctatactg atttgaagtg 2034gagctgttca gaactgaacc ttgtaggaaa ttccaagggc ctttctactg aatctggtga 2094tggggtgggg ccgtggcact ttctctgcca cagctgttct tcacagtgtt ggtgctaatg 2154aggccagggt gcagggttcg attcacacgt aggccagtta acttagagaa aatctatttc 2214cttacctcta gccagtcact tcctttttcc gcagttgtga tgggttttgc tgagccatcc 2274actctgactg atttcctctg aagtaaacat atttacaatc caaagcaatt ctactgacag 2334aagtgttgcc ttcataatca aacagcttgt ttttccatct cctctgcaac cctaattaaa 2394tgagtacagg tctacaaaat gttttcaagg agaaaagcag catatcctta agtgaagtat 2454tatatttttc aataaccctg tagtggcttg atgcagggaa ccctggggga ctttcagcga 2514agagctgtgc tcttttctga ctagattaga gcgtttggag tggaagacgt caaatgtgta 2574gtgagatgga ggttttacat tgttcttcta ctggctgtga tgaagtgcca gaatgtctct 2634ttagaacaag agttagattc cccctttctc cttattgccc cttccgtttt gacttcccct 2694ttatttattt gctgtctaat taggggccaa gtctgtaaag ttttgtcaaa gtgagttaga 2754agttgttttt tcttactatt tgtgtttacc agagttggga gataagatag tttccatgaa 2814ggtgtgtatg ttttatacga tgtttgttat agggccatgc attggtaact tgaaaataag 2874acagcttaat gtcttcagga tgtaatcaga cagcttaatg tcttcaggga tgtaaaactc 2934tgactacacg gcgtctcttt ttcatacatt gcatgtaagt tgttagtacc tcacaagcta 2994cagaagttca gccatgagat tttgtttggc aacatgaaca gatttgtgta taactgcaat 3054ggcctttttt ccagatttcc ttattgactt tttgtttgcc ttacctgggg ctagtttttt 3114atgctttgta cctagaaaac aaaaaattac attcgttggg ctttttttca aggttgggat 3174taccacacca cctggaatat catactgtgg tttctgccta aaattggcac atgtaagtat 3234tgaagaaaat ggttatataa ttcagttgaa actcttggtt attagatgtt aggcatctcc 3294tgtatgtaag acacaaggcc aaccacaaca cagaacgatg ttgacctgtt aagtattctc 3354tgaaacatgg ccaaaatgca ttttatgagc tttttttttt tgctattgta aatattagtg 3414gtttacaatg cgctttaaac atatttcttt aaaatgcaag cagtgagaaa taagacctct 3474ctgaattagt agctctaaac tgttaacata gaatgttact tggaaaaagt ctggaatatg 3534tggtgtacac aagcagtgct tcgtgaatga gtttcttagc ttttatagtg cgccatgttt 3594ctcaaagttt gtttttgttg acaaaacatt ttataatata tatcttatgt ttattttttt 3654tctcaactaa ttgtgtactg cactgtaagg tgaaaattag ccatccatta tttatcttct 3714gtggcaatgc atttatatgg ttgattgggt ggggaatttt ttgcagaaag atgcaaagtg 3774atgggttttc gacttcctat cgcagggagc ttttaagaaa tattaatttc ctatacattt 3834ttccaatccc catgcaaact gttcctgttt acataccttc tctgttgtat cagtactttg 3894agtgagaaga cagtttattt aaaacttgag caggctgttc agcatttttt ctgcttctga 3954aatctgtata gtacactggt ttgtaatcat tatgtcttca ttgaaatcct tgctacttct 4014cttcctcctc aatgaaatac attatatatt atctttatgt actcttaaga aaaacgagca 4074aggaagagta tcttcattat tctcattttc tctgagttgg aaacaaaaac atgaaggact 4134ccaactagaa gacagatatt tacatttaaa tagattagtg ggaaaacttt aagagtttcc 4194acatattagt tttcattttt tgagtcaaga gactgctcct tgtactggga gacactagta 4254gtatatgttt gtaatgttac tttaaaatta tctttttatt ttataaggcc cataaatact 4314ggttaaactc tgttaaaagt gggccttcta tcttggatgg tttcactgcc atcagccatg 4374ctgatatatt agaaatggca tccctatcta cttactttaa tgcttaaaat tatacataaa 4434atgctttatt tagaaaacct acatgataca gtggtgtcag ccttgccatg tatcagtttc 4494acttgaaatt tgagaccaat taaatttcaa ctgtttaggg tggagaaaga ggtactggaa 4554aacatgcaga tgaggatatc ttttatgtgc aacagtatcc tttgcatggg aggagagtta 4614ctcttgaaag gcaggcagct taagtggaca atgttttgta tatagttgag aattttacga 4674cacttttaaa aattgtgtaa ttgttaaatg tccagttttg ctctgttttg cctgaagttt 4734tagtatttgt tttctaggtg gacctctgaa aaccaaacca gtacctgggg aggttagatg 4794tgtgtttcag gcttggagtg tatgagtggt tttgcttgta ttttcctcca gagattttga 4854actttaataa ttgcgtgtgt gttttttttt tttttaagtg gctttgtttt tttttctcaa 4914gtaaaattgt gaacatattt cctttatagg ggcagggcat gagttaggga gactgaagag 4974tattgtagac tgtacatgtg ccttcttaat gtgtttctcg acacattttt tttcagtaac 5034ttgaaaattc aaaagggaca tttggttagg ttactgtaca tcaatctatg cataaatggc 5094agcttgtttt cttgagccac ggtctaaatt ttgtttttat agaaattttt tatactgatt 5154ggttcataga tggtcagttt tgtacacaga ctgaacaata
cagcactttg ccaaaaatga 5214gtgtagcatt gtttaaacat tgtgtgttaa cacctgttct ttgtaattgg gttgtggtgc 5274attttgcact acctggagtt acagttttca atctgtcagt aaataaagtg tcctttaact 5334tcaaaaaaaa aaaaaaa 53514164PRTHomo sapiens 4Met Phe Gln Arg Leu Asn Lys Met Phe Val Gly Glu Val Ser Ser Ser 1 5 10 15Ser Asn Gln Glu Pro Glu Phe Asn Glu Lys Glu Asp Asp Glu Trp Ile 20 25 30Leu Val Asp Phe Ile Asp Thr Cys Thr Gly Phe Ser Ala Glu Glu Glu 35 40 45Glu Glu Glu Glu Asp Ile Ser Glu Glu Ser Pro Thr Glu His Pro Ser 50 55 60Val Phe Ser Cys Leu Pro Ala Ser Leu Glu Cys Leu Ala Asp Thr Ser 65 70 75 80Asp Ser Cys Phe Leu Gln Phe Glu Ser Cys Pro Met Glu Glu Ser Trp 85 90 95Phe Ile Thr Pro Pro Pro Cys Phe Thr Ala Gly Gly Leu Thr Thr Ile 100 105 110Lys Val Glu Thr Ser Pro Met Glu Asn Leu Leu Ile Glu His Pro Ser 115 120 125Met Ser Val Tyr Ala Val His Asn Ser Cys Pro Gly Leu Ser Glu Ala 130 135 140Thr Arg Gly Thr Asp Glu Leu His Ser Pro Ser Ser Pro Arg Ala Arg145 150 155 160Lys Ser Cys Leu517518DNAHomo sapiens/DEFINITION=Homo sapiens p53DINP1 gene for p53DINP1a, p53DINP1b, complete cds, alternative splicing. 5tcatagctta ctgcatcctt gaacctggat tcaagagatc ctcccacgtc agcctcccaa 60gtagctagga ctacaggtgc acaccactgt acctggctaa ttttttaaaa tttttttgta 120gaaacaggtt attttgttgc ctaggctggt ctccaactcc taggctcaag cattcctgtg 180gcctcagcct cccaaaatgc taggattgca gacatgagcc tccacaccta gcctcatcca 240ttcctatgct ttttatcact gtctttgtgt catcagctcc caaatctatg cccctagctg 300agaccacact cctgagctcc aaagctctat atcctgctgc ctgctggaca tctccaactg 360cattattttg catatccccc caatttacca tattcgaaat gcctaacctg tactttctgc 420aggattcatt cctgtttccc taaatggttc caccatcctc tcagttggcc aagccagaaa 480catggggtnt gagacttttc cccttcccct cttccaggct taacaaggcc ctgcaggacc 540tgttcctgct cagctcccct agcctgctat tgcgaccctc cctctggacc ttgagcggta 600ctgaaggtgc cacattctca ctcatccaca gggttccata gtgttcattt gcccccttcc 660tccagcactg acctcagcca ctcaagagta aagtcattgc aggagtccac acaggtagcc 720tggagaacac caaatacttc ttccatggca ttcatcacac cggattgtta aatttgtggg 780agggccctgt tcacagccat ctcccctgtg cccagcaatg cacctgatgc ataacaggtg 840ctcagtaaat acatgtagaa taatagttga cctggactga ggactttatt cgaagtattt 900tgctaagtcc atcgccttta acaagagtag gatcagggag actgtctagt agacagtttt 960ggcaaagtct cagagacaac agtttggctt aaactaggat gagagtggtg gatggaagca 1020ggcagattcg agctgcctgt gctcctgggg ttgacgggac ttggtggttg ggatgagggg 1080tgagtaggag ggagcaaaag gcatgcgggt gcggcagctc acgcctgtaa tcctagcatt 1140ttgggaggct gaggtgggcg gatcatgagg tcaggagatc gagaccatcc tggctaacat 1200ggtgaaaccc tgtctccact aaataaaaaa aaaagaaatt agccggatgt ggtggtgggc 1260gcctgtagtc ccagctactt gggaggctga ggcaggagaa tggcgtgaac ccgggaggca 1320gagcttggaa tgagccaaga tcgcctcact gcatgcactc cagcctgggc aacagacaaa 1380gactccgtct caaaaaaaaa aaagcgtgaa gggtgactgc tagggtgttg gcctgaataa 1440ctgccagctg ctgctgcttc ctgagaaagg gatgaagggc agtgtatatg aataagtttg 1500tgggggtaaa aaacctatct ttggtatgtg tttggtttga tacgcccatc aggtagccag 1560gtagaaatgg ataaatattc caagatcaag caagcgtgga gtttttcatt tagaactcac 1620cagaattccg taaggtagga gctgttgcca gctgaagtca cataacttcc taactcacac 1680agcaagtgag cagctagcca gggtttgaat gcaggtggct gacccagcac ccacctgctg 1740agctgctaca caggaagggt gtttccagtt ctaggtttgg ccaagctcag tttcctacag 1800tgtaaatttc ttcatgccag ctgtaggaat tgtttagaag gtcaaagtat gcttgctggg 1860cttacagatt ttaagtgtaa tcaggtgtgc ttacagttat agacaagaag tacagagtag 1920aagagaaaat gagccagagg aggatgaggg cctggcataa tggtggtcac caggcactgt 1980tgctgggact tctgagcagt tgaataacat atagcaagac tgttactatc tagaatttgc 2040tcatctgctt gcagaaaagt ttgaaggata ttattcaaat tcaccttatt ataaaagcat 2100aaatatgaaa aagaaccaac tctttccctt ttatttcaca ttttaagata tttataggtt 2160gaatttgagt aagagtattt acaaaacatt atgttgcatt tcttgtgttt gactcctgca 2220gaaagggaat agctgtagaa tgtaggccaa ggcatatgct ttcttctaag ggtatgtgtg 2280tatgtgtatg tacttatgta tatatgtgca tgctgatttg aaataacttc ctttttctgt 2340tttattttag gtacaatgac tcttcttgct tttcacctaa gttgaataag caccctgtgc 2400actttaatct cctgtcggta ccattgggcc aactaaagac aaggttttga aatctcagct 2460ataaaagaca tccagccaaa ctctcagtct tgccttaaca atgttccaga ggctgaataa 2520aatgtttgtg ggtgaagtca gttcttcctc caaccaagaa ccagaattca atgagaaaga 2580agatgatgaa tggattcttg ttgacttcat aggtanggta cactnggtta ctnggtggtn 2640canctncaca gtaaaaatgg aaacttgcnt ctcanttttn gcatanggaa accaataact 2700gaggggtttn canccttgaa aatgaagcag gatttgtctt taaaaaattg caggaaacat 2760ctgtgctaga taatgtcagt gaaattgact gacatcagac tttttagttt tgcaactctc 2820acagattgat tagatcaagg gcacttcatt agaagcccct tttattagac aagtagtaac 2880caatatgtct cattacatag ttatccagca gaatagaaac agtcttaaaa tcactcctgc 2940aaagccaaca taaatctatc ccatatagtg acacacacac acacacacag aatttctgtg 3000ctcagaaacc catttatagt catatagatg aatataaatg ggtttctgaa ctaaataaaa 3060gaagatacgt gagcatcagc taatagtatc atctcttctt tttctgtgct gtgaatttga 3120tgggtgactt tataatgact gcccaatgtg ataatcatcc tcctgtggga ctcttcaaca 3180acacacgtgg actgcaatgt ggactacgat ctagatactt gcactggttt ctcagcagaa 3240gaagaagaag aagaggagga catcagtgaa gagtcaccta ctgagcaccc ttcagtcttt 3300tcctgtttac cggcatctct tgagtgcttg gctgatacaa gtgattcctg ctttctccag 3360tttgagtcat gtccaatgga ggagagctgg tttatcaccc cacccccatg ttttactgca 3420ggtggattaa ccactatcaa ggtggaaaca agtcctatgg aaaaccttct cattgaacat 3480cccagcatgt ctgtctatgc tgtgcataac tcctgccctg gtctcagtga ggccacccgt 3540gggactgatg aattacatag cccaagtagt cccaggtacg ttacaagtat ttaactctct 3600gtaggcagtc cactgagcat gcattttgtc tctactgtaa ttcaaagcta agttattaga 3660agatgcaaaa ctaattttgt tagagattta tgaaagcatg tgcatagtac acaagatggt 3720ccgtaagata acttgttgac cacagtgtct cacacctgta atcccancac tttgggaggc 3780caaggcaggc agatcactta agctcaggag ttcaagacca ccctgggcaa catggtgaaa 3840ccccgtctgt ataaaaaata acaaaaaatt agccaggtgt ggtggtgcac acctgtagtc 3900ccagctactc aggaggctga ggtgggagga tggcttgaac caggaggcag aggttgcatt 3960gagtcaagat tgtgccactg cacatcagcc tgggtgacag agcaagactc tgtctcaaaa 4020acaaaaactt gttcatttta gattttttta agaaagttag atgtagcatg gccccagtta 4080tataatagca ttctcattta caaaattctg ctactgatta gaaataanac tggcatttct 4140gttaagtatc ttanaacata actctgactt tatagactgg attatttacg gtatcaaatt 4200caactanaat atcatntctt aaaatttatt ttcttccaan tatttctanc tacatctcat 4260caaatgatgt anccttgaaa accttgtctg agaatttaac aaactcantt tcttcctgca 4320atactcacac tgcacagtca cccantgctt ctgacaccan atgtgtnggt tttttccacn 4380cccangcaaa cagtccccat cagtggacac cctctaantg tcctctgatt caattcaatt 4440ctgacactgt ctacctccan attgcatctn aatccatagg ttaaaggctt agtcccaaaa 4500gactgcctcc catttctgat gccaatctca agccccaggt tttacttatg cttctgacca 4560accagctgta aactggtgtt cccatgaccc cttccttggg tttgattaat ttgctanaac 4620atcttatana acttggggga acatgtttac cantttatta aaaaggatac agatgaaggg 4680atgcataagg caaggcccat gccctctgtg caccccaccc tccgggaacc tccacatgtt 4740canctctcca gaagctccct gaactcanta ctattggggt cttatggang catcngtaca 4800taggcataat tgattaaatc attggccatt ggtgattggc ttaaccttca aacccnctcc 4860cctctctgga atttganggg tngggctaaa antcccaact gttacttggt ctttatagtg 4920accacatccc atcctgaanc tgcctaattg ctgccaacca ccagtcaact aattagcata 4980cgctttccac attggaggtt ccaagggatt ttaggagttg tatgccagga aactgacaag 5040accgaatata tattttacag tatcacagtc cagcctcagt cttcaaactt ggattactta 5100caacaaaagg atatggaact caaaaaatac tggcacaaca ccagaatccc attcagtcag 5160taattcattc catccatcat cgtactgtat gagcatgtct cccagggtga ggccactcag 5220gtttgcaggt taattgatct tgttggtttc caaaaccaga aatggtttta gcaaacacgt 5280agcttcaccc tttcaggcat ctggtatacc tgagcttana aacaatgtca tctttttctc 5340tgancctgtt tcaaggtatt aatataatga attgatccca attcataacc tatttattca 5400ttcttttgtc ttctttctct tcatttatac ccaaactttt ccatctctgg aanggacgtt 5460agctttancc actgtgctgg tctanattgc aggtagcaat actagtttag caagtacctc 5520tccctcagtc cactcccatt cagatagggt taggtgacat aggtacaaaa ttagtaggct 5580gtttttacca ccagacaata cagctgcatt cgttgttggc tccanttttg caagatgggg 5640tctaggtaac ccccataana cccttaggaa ttttgacata aggtttaaaa acatatttac 5700agcttcttgc tcaggaacca tccctgcttc cagtacttgt agttacancc ctggtcctan 5760gaccattgca tccagtacan gaaaaaaaaa tgangtgatg tgggaaacaa aaanttaaag 5820tcattatact ancatactta acccttggct acaattgcat aantcatgcc ancatcctcc 5880tccccatccc ttcttcctca gataccanaa aaacangaaa atcttgtggg gacatccaac 5940ccttcatnct tttatccccc cantttgttt ttgggttttt tttttttttt gaaataaant 6000ctcactgtgt cacccangct ggagtgcant ggcaggatct tggctcagcg caacctctgc 6060ctcccgggtt caantgattc tcctttctca gcctcccaag tagctgggat tacaggcgcc 6120cgccaccata cctgggtaat atttgtactt ttagtanaaa gggggtttca ccatatttgc 6180cangctantc tcnaactcct gacctttggt gatccaccgc ctctgcctcc caaantgttt 6240ttgggcatta tgggctgtat aatgtgtcct ttggtctgaa aaaatgacan ttaactatcc 6300acatccaggc agtatcttct gttgtangtt tttttggcac tctgagcatt tgcatcttcc 6360attggttaag taaagcccac tccagagact gtcttttctt ctcaagaccc attttgtagc 6420tccccaggac taccagcact agtctcactt gcagctatgc tcagggcctt cccaccaggg 6480gaatctgccc ctcatcagta gtctgtctct tttgctggct ggcagaacgg ttctcattgg 6540tatttgtgcc tgagagagta gaagaggaat gtctagatgc agcctgtctc tgcactgcct 6600gtaccactca tttaatgaac ccaggatggc acctccaggg gagcacatgg agatatatct 6660gcttccagtc ccagtcacct tccaaacctg gaanggagtt cttctgatgg ccactggcat 6720gtcctaatat acccctcaaa tttccgtagg gttgtgctcc ctatggaagc catcccttta 6780aaantccagg ttttcattgt gattctgcct gaccatgtcc actanccact gcccatgant 6840cagtaaaaac caaacctagg ggcttccatc atcgctanga aaacagcgtg caattcancc 6900ccncanaacc gaattgtttt taccttcttt gatcaaagta gcatccttcc aaacaagatg 6960ctgtctgttc acgttggaac tgccatccac aaccaagcag ctctttgtca gtcagttgan 7020anctattggc agtgtggaat ccagctcctc acacagttcc agantcagtc caaggtgggg 7080aaangctccc tgcttgtggg tgtgtccttc ttacattccc tacataacat gatcctgtat 7140aaaccatttc catttattat ggaactcttc tgggcactgc cctcctaatt agtgtttctc 7200tgacatcacc aaacacagca tgggtatttc aggtttcaan atcattttat gtccttcaat 7260cctaggggta gcttcaatta atgtcctgtg gcaanggant aaatgcctct catgtggaaa 7320ttctctagtc caaagtgcna ntaattgtta ctganaagtg ttcacagtct tttgccataa 7380gtgccagttt cccttccaca taagactatc agacagataa tttgtcccta ccaccactcc 7440agcttctact atacaccatt gggcctgggc agtgctgttg gtccccattt agcatgtcca 7500gttaatattg tcatgaaggg cagatttaca tgccttttgt tttacagtac tangtanggg 7560aagtgtgtct ccagccagac atatccattc tcataaaaca tttaggtaaa ggggatataa 7620cttctttacg taaagctcac ttaaacatcc taactttcat aattctatca acccttgcat 7680ttttatgttc tccaatctaa ttgtatcctg aatcagggct ttaccaatag atcttgatac 7740cacantacat ggagctccca taccaangan tcccaggaat ttatcttcac caccccgacc 7800attttaccca cttgtntatg anaagccttt ggttcccnnc acaaacagaa aaatanatcc 7860ttgcccattt gtcagtcatt atcttgantt aatcaatctg tctattgccc caagcaattg 7920ttggtcactt ttcttgtaat ctctgccttc tggcttttta agtttccccg aatttacaga 7980catcatatga cctttgtcca nagaatatag ctaggaaata tctganattg ctcccaccca 8040ctaaagtaaa ccccattacc cagttaatta nctgaaagtc acactgttct atcgtntacc 8100ttagtngaat ntttcctgtt aaantatntn gggaatgttg gcattccctc tcttaagttn 8160agggtccaan tggatttnga tccttggagc acttcncaan atccctattg tttntgttgt 8220catgtccatc nctctgactc taanaaaaat attgtattat atctatctat ctatctatct 8280atctatctat ctatctatct atctatctat ctatatctat ctacctatgt acctacctac 8340ctacctacct acctaccgtt tcctgttttc cagatataaa caaaccctag gcagcacatt 8400aatttattaa ctttaccaga gtttgccata ctctcaagtc tcaatattgt agatgatttt 8460ttatgaggct aaacatattt cattttgctt gtaccaaaat gaaaacaact ttcttatgtt 8520acatgatgcc aggctatatc ctatgtaagg tcatgggtgg ggaatnttac tttaactggc 8580actttattca gtgctacaag tttattaaga ngtgtttcag gcattccaac aaaaatttaa 8640aatataactt tgtggagttg cnattgattt aattgcttta gagttgcttc agattcattt 8700cttgaaaatt cttcccttct gattatgatt ttacctctga agaatgccac tcaagaattt 8760atnattangc caggtgcagt ggcttaccct gtaatccaag cactttggga ggctggggtg 8820ggtggatcac gaggtcagga gttcaagacc agcctggaca acatggcaaa accccatctt 8880aaaaatacaa aaattagcca ggcttgctgg tgggcgcctg tactcccagc tactagggag 8940gctgaggcag gagaattgct tgaacccggg aggcagaggt tgcagtgagc tgacatcacn 9000ccattgcact ccancctggg tgacaaanca aggctctgtc tcnaaaaaaa aaaaaataga 9060atttataatt aatagtcact ttaaacttac tggcatttat atgtttactt tgcagtgttg 9120tgtgtgtgtg tagtcagcag ctttgcaaaa gaatttacta cagaaacagt acaaatgcaa 9180tgtaaaaaaa ttagaaaata caaatatgca aaaattggaa atgacatttt catgacccan 9240anatnaatac ttttaacatc ctanagtata tccttccana ttgtgttttt ttttttctgt 9300gcctgtacgt acacataaac ttttaaccta aatggcaaca tgccatatat acttcttttt 9360ttttttttga nanaaanttt cacncttgtt gcccaggctg gagtacaatg ttgcaatctt 9420ggctcattgc aacctcttgc ctcccgggtt caagcaattc tcctgcctca gcctcccaag 9480tagctgggat tacaggcatg tgccaccgcc actacgccca gctaattttt gcatttttag 9540tagagacggg gcttcaccat gttggccagg ctggtcttga actcctgacc tcgtgatcct 9600cccccccccc ccacaaagtg ctgggattac aggcatgagc caccatgccc agatgtatag 9660ttggtttttt tgggggtttt ttttgaaaaa atcttgctct gtcacccagg ctggaatgca 9720ttggcgcgat ctcagctcac tgcaacctct gcctcccaaa ttcaagcaat tctcatgcct 9780cagcctcccg attagctggg actacaggcg tgcactacca cacctggcta attgttgtgt 9840ttttagtaga gacagggttt caccatgttg attaagctgg tctcaaactc ctgacttcag 9900gtgagccacc tgccttggcc ttccaaagtg ctgggattac aggtatgagc cactgtgccc 9960ggcccagata tactatttta taaactactt atttaggcaa tgatntccac cattttgttt 10020cagtagattt cttcttcttt cttccttctt cttccttcct tctttttcaa ggttgttatg 10080gttttctagt tttggtacag tgagaatgtt gtttttatat cttgtttgtt tttgcattca 10140tatatgatag ttatataggg atagtaaaac aaatacatac ttttccatat ataaagggtc 10200aaaggaaggc actgaatttt atttatgtat tttaacacgg agtntcactg ttgcccaggc 10260tggattgcag cggcgcaatc tcggctcact gcaacctccg cantgggttc aagtgattct 10320cctgccttat cctgccgatt aactgggatt actggcacat gccaccatct ccagctaatt 10380ttttgtattt taagtaaaga tggggtttca ccatattggc caggctggtc tcaaactcct 10440gacctcaagt gatccgccca ccttgacctc ccaaagttat gggattacag tcatgagcca 10500ttgtgntcgg ccagaatgca ctgaatttta tgtgatcctt tttgtacaca ggaaactcct 10560gtaaattatt agttttaaaa tagtaactat aattttttta acatccttat tttgggattt 10620gaaattcttt tttttttatt tttgagacgg agtctcgctc cttcgcccag gctggagtgc 10680agtggcgcta tctcggctca ctgcaagctc cgcctcctgg gttcatgccg ttctcctgcc 10740tcagcctccc gagtagctgg gactacaggc gcccgccacc gcacccagct aattttttgt 10800atttttagta gagacggggt ttcactgttt tagccaggat ggtnttgatn tcctgacntc 10860angatccgcc cgcntcagct tcccaaagtg ctgggatnac aggcgtgagc caccgtgccc 10920ggcagggatt tgaaattctt ataaggaagt cttgctaaat caaggtggaa atcaggtgtt 10980accaaaatta ggctagcttt gtagtaaata tntgttttnt tagcaggaaa tgaatcaaat 11040gtctttcaga agtcagattg cttccattgt gtctatcatt ggggatatta ccataaagca 11100cagaaaattg aggtgctttg taattacagt gggcttcagg gtatacattt cattgcattt 11160tagcctttat aagtattgtt tttgaaggtt ttacatgtta tcttaagcac atgtacgtgc 11220ctttaatggc acatagtaaa ccagttttaa taaaagttga ccacgtccta atagtaaagt 11280tacaatagca ttttaaatct tactagtgat ttaatagttt caccatttaa catttgtagg 11340gccaggaaaa gctgcttata agactcacgg gcacagagta agtatattca ctttgagact 11400gtgtgatata tgtatgcata gggaggcaaa ggacccaaat gctgaccttt taaaaatgac 11460tttctttctt tttttganat ggantctcac tctgtcactc aggctggant gcaatggtgt 11520gatattggct cacagcagcc tccacccccc aaattcaant gattctcttg tttcagcctc 11580ccaantacct gggattacag gcttgcatca ccacacctgg ctaatttttg tatttttagt 11640aaaaatgggg tttcaccatg ttggccagnc tggtcttgaa ctctattaan gtgatccacc 11700tgcctcagcc tcccaaantg ttgggattac aagancganc cacagcgcct ggcctaaaaa 11760taattttctt tatatnaact tttttctatt antttcttat tgtggcctct taaaatncta 11820ctgtaaaaaa attttatata tatatgtgtg tgtgtgtgtg tgtaataaat atgtgtgttt 11880cctcaagtga ttttaggcag aatgtgtcac tgcccttcct gtgtaaaata actcttaaaa 11940atcatacagc tcttcatgtt acgtgttgac atagtttaca gcaatcctac ttgattagct 12000tttttatatg gaaaacataa tcatactgtg tgtagccaat tttaagattg tgttattcaa 12060gantcttata ttgccatgta aataccatct caaaatgtgc cctgttgtga gataaagaaa 12120aacttaagag atttattcta ttaagctata gctttttatc tagaaaaatg gcaaatgagt 12180agaatttcca tatgcaaatt tctattaaat aaagatttaa agcatgtttc ctggaacatt 12240ttacttatag acaggaaatg tcttgtgaag gtattgtgag cacactgctt tctgatcagt 12300aaaattnggg aattaaaata ttttcctttt aagattatgg tgagaattaa aatatatatt 12360tgcaaaagaa ttggcaaatg cagatattca ataaatgtta gntcccctcc cagacaagct 12420tatataaatg tgtgtgctcc gtaaatgaaa ttcaaagttt aaagtgtatt tttagaaaac 12480agtctactta gtgagccatc tggggcttat atggctcttt ctataacagt tgtttccttc 12540caccactatc agtcagtggg gtnanngaat aataaaattg tttgtnagcc atgaaacana 12600agntttnatt nagccataaa caaantagng tggcaaattg aattgntaag taacttttcc 12660agttttgtta ataacatant ctgttattta anacatcccc accccnttct gtttcagagt 12720ggaagctcaa aatgaaatgg ggcagcatat tcattgttat gttgcagctc ttgctgctca 12780tacaactttt ctggaacaac ccaagagctt tcgcccttcc cagtggataa aagaacacag 12840tgaaagacag cctcttaaca gaaatagcct tcgtcgccaa aatcttacca gggattgcca 12900ccctcggcaa gtcaagcaca atggctgggt tgttcatcag ccctgcccgc gtcagtacaa 12960ttactaatag tttcaagttt tgttggttgg tttctcttgg tttgtgctaa catgtatgga 13020tgtgtgtata tgtacagtga aaatgttgtc tctttacaac caattgataa ccaatcacat 13080agttttatca gtgtatttag acactatctt gaaaatcaga tttatatgct gtgtatcaca 13140taatgccttg cctttaacat ttactttttt tgtacacttt ttcagattat ttctggaaac 13200atatcaatat aattacagtg tttgggggtg tctttaaata tattaggtta tacattagtc 13260agcattttaa agacatttct tcccaagtac gagaataggc atctttcatt ttcattttat 13320tttgtattac ttaatctttt aagcaagcaa aaatttattc tcagggtcag ctgtacactt 13380tattgaccag tacttgataa tctctctgta tatgatgaat acatttttat acactaacat 13440tagcattaac aggtgatagt tgccatggat ataatggaat tatggctgga ctttcttttg 13500aaagaaaact tgatgtattc tgtgtgtatg gtttttcccc agattagtca tacagttcat 13560ttggaattca ggtacattaa gctttagtga agagtgcatg cagtaattcc aatgtgactg 13620catgacgtgg tacagacatt acaggtgttg tagacagagg cacttgtctc gtgcagaggg 13680attaaattag acctgtgaga ttatatttgg aaaaattcat gtctgtaact aacccattag
13740tgcagtattt aatttgttac tattccttcc cgccaattct gtccactcct cacctcgcat 13800cagctataaa tttggaaata cttgtccagg cactcaagtg acttcatatt tctctctgcc 13860catgggaaaa gagataggct ttatatttcc acagantgaa aaatcctctg tcatggagcc 13920tgtcctgcca agtggcaaga atgtggggac tgtctggtga tgatgtcttt catggcatct 13980gagtgaagaa tgacaggttg gctcaacttt tttctttttt ttttttaatt gccttgtatt 14040gtaagtattc ttccctgcag tccaagtgac ttttcatttt ttgttttaac ttcaggcaaa 14100atctttaacc actctggcct ctgtttcccc caccaacggg gagcagtgac atttacctcc 14160ctcacagagt cactgtgagg attctatact gatttgaagt ggagctgttc agaactgaac 14220cttgtaggaa attccaaggg cctttctact gaatctggtg atggggtggg gccgtggcac 14280tttctctgcc acagctgttc ttcacagtgt tggtgctaat gaggccaggg tgcagggttc 14340gattcacacg taggccagtt aacttagaga aaatctattt ccttacctct agccagtcac 14400ttcctttttc cgcagttgtg atgggttttg ctgagccatc cactctgact gatttcctct 14460gaagtaaaca tatttacaat ccaaagcaat tctactgaca gaagtgttgc cttcataatc 14520aaacagcttg tttttccatc tcctctgcaa ccctaattaa atgagtacag gtctacaaaa 14580tgttttcaag gagaaaagca gcatatcctt aagtgaagta ttatattttt caataaccct 14640gtagtggctt gatgcaggga accctggggg actttcagcg aagagctgtg ctcttttctg 14700actagattag agcgtttgga gtggaagacg tcaaatgtgt agtgagatgg aggttttaca 14760ttgttcttct actggctgtg atgaagtgcc agaatgtctc tttagaacaa gagttagatt 14820ccccctttct ccttattgcc ccttccgttt tgacttcccc tttatttatt tgctgtctaa 14880ttaggggcca agtctgtaaa gttttgtcaa agtgagttag aagttgtttt ttcttactat 14940ttgtgtttac cagagttggg agataagata gtttccatga aggtgtgtat gttttatacg 15000atgtttgtta tagggccatg cattggtaac ttgaaaataa gacagcttaa tgtcttcagg 15060atgtaatcag acagcttaat gtcttcaggg atgtaaaact ctgactacac ggcgtctctt 15120tttcatacat tgcatgtaag ttgttagtac ctcacaagct acagaagttc agccatgaga 15180ttttgtttgg caacatgaac agatttgtgt ataactgcaa tggccttttt tccagatttc 15240cttattgact ttttgtttgc cttacctggg gctagttttt tatgctttgt acctagaaaa 15300caaaaaatta cattcgttgg gctttttttc aaggttggga ttaccacacc acctggaata 15360tcatactgtg gtttctgcct aaaattggca catgtaagta ttgaagaaaa tggttatata 15420attcagttga aactcttggt tattagatgt taggcatctc ctgtatgtaa gacacaaggc 15480caaccacaac acagaacgat gttgacctgt taagtattct ctgaaacatg gccaaaatgc 15540attttatgag cttttttttt ttgctattgt aaatattagt ggtttacaat gcgctttaaa 15600catatttctt taaaatgcaa gcagtgagaa ataagacctc tctgaattag tagctctaaa 15660ctgttaacat agaatgttac ttggaaaaag tctggaatat gtggtgtaca caagcagtgc 15720ttcgtgaatg agtttcttag cttttatagt gcgccatgtt tctcaaagtt tgtttttgtt 15780gacaaaacat tttataatat atatcttatg tttatttttt ttctcaacta attgtgtact 15840gcactgtaag gtgaaaatta gccatccatt atttatcttc tgtggcaatg catttatatg 15900gttgattggg tggggaattt tttgcagaaa gatgcaaagt gatgggtttt cgacttccta 15960tcgcagggag cttttaagaa atattaattt cctatacatt tttccaatcc ccatgcaaac 16020tgttcctgtt tacatacctt ctctgttgta tcagtacttt gagtgagaag acagtttatt 16080taaaacttga gcaggctgtt cagcattttt tctgcttctg aaatctgtat agtacactgg 16140tttgtaatca ttatgtcttc attgaaatcc ttgctacttc tcttcctcct caatgaaata 16200cattatatat tatctttatg tactcttaag aaaaacgagc aaggaagagt atcttcatta 16260ttctcatttt ctctgagttg gaaacaaaaa catgaaggac tccaactaga agacagatat 16320ttacatttaa atagattagt gggaaaactt taagagtttc cacatattag ttttcatttt 16380ttgagtcaag agactgctcc ttgtactggg agacactagt agtatatgtt tgtaatgtta 16440ctttaaaatt atctttttat tttataaggc ccataaatac tggttaaact ctgttaaaag 16500tgggccttct atcttggatg gtttcactgc catcagccat gctgatatat tagaaatggc 16560atccctatct acttacttta atgcttaaaa ttatacataa aatgctttat ttagaaaacc 16620tacatgatac agtggtgtca gccttgccat gtatcagttt cacttgaaat ttgagaccaa 16680ttaaatttca actgtttagg gtggagaaag aggtactgga aaacatgcag atgaggatat 16740cttttatgtg caacagtatc ctttgcatgg gaggagagtt actcttgaaa ggcaggcagc 16800ttaagtggac aatgttttgt atatagttga gaattttacg acacttttaa aaattgtgta 16860attgttaaat gtccagtttt gctctgtttt gcctgaagtt ttagtatttg ttttctaggt 16920ggacctctga aaaccaaacc agtacctggg gaggttagat gtgtgtttca ggcttggagt 16980gtatgagtgg ttttgcttgt attttcctcc agagattttg aactttaata attgcgtgtg 17040tgtttttttt ttttttaagt ggctttgttt ttttttctca agtaaaattg tgaacatatt 17100tcctttatag gggcagggca tgagttaggg agactgaaga gtattgtaga ctgtacatgt 17160gccttcttaa tgtgtttctc gacacatttt ttttcagtaa cttgaaaatt caaaagggac 17220atttggttag gttactgtac atcaatctat gcataaatgg cagcttgttt tcttgagcca 17280cggtctaaat tttgttttta tagaaatttt ttatactgat tggttcatag atggtcagtt 17340ttgtacacag actgaacaat acagcacttt gccaaaaatg agtgtagcat tgtttaaaca 17400ttgtgtgtta acacctgttc tttgtaattg ggttgtggtg cattttgcac tacctggagt 17460tacagttttc aatctgtcag taaataaagt gtcctttaac ttcaaaaaaa aaaaaaaa 17518621DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized primer sequence 6ttgtgggtga agtcagttct t 21719DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized primer sequence 7gagcttccac tctgggact 19820DNAArtificial SequenceDescription of Artificial Sequencep53-binding sequence BS in intron 2 8gaacttgggg gaacatgttt 20929DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized primer sequence 9cgccgagctc cctgcaatac tcacactgc 291029DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized primer sequence 10cagtacgcgt cctccataag accccaata 291122DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized sequence 11cgaacttggg ggaacatgtt ta 221230DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized sequence 12cgcgtaaaca tgttccccca agttcgagct 301342DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized sequence 13cgaacttggg ggaacatgtt tgaacttggg ggaacatgtt ta 421450DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized sequence 14cgcgtaaaca tgttccccca agttcaaaca tgttccccca agttcgagct 501516DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized antisense oligonucleotide sequence 15tggaacattg ttaagg 161616DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized antisense oligonucleotide sequence 16tcagcctctg gaacat 161716DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized sense oligonucleotide sequence 17ccttaacaat gttcca 161816DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized sense oligonucleotide sequence 18atgttccaga ggctga 161920DNAArtificial SequenceDescription of Artificial Sequencean artificially synthesized nucleotide sequence 19rrrcwwgyyy rrrcwwgyyy 20
Patent applications by Hirofumi Arakawa, Tokyo JP
Patent applications by Yusuke Nakamura, Yokohama-Shi JP
Patent applications by Oncotherapy Science, Inc.
Patent applications in class Tumor cell or cancer cell
Patent applications in all subclasses Tumor cell or cancer cell