KR20040063422A - Apoptotic inducer containing deleted proteins of CDC6 - Google Patents

Abstract

PURPOSE: Provided is an apoptosis inducing protein which controls the death of the cells to prevent the diseases due to the pathological growth of those cells, and particularly, inhibits the cancer cells. CONSTITUTION: The apoptosis inducing protein comprises amino acid sequences of 1-179 of the CDC6 protein identified by SEQ ID NO 1 where the amino acid sequences of 462-488 are deleted. The protein additionally comprises the amino acid sequences of 180-442 partially or wholly.

Description

Apoptotic inducer containing deleted proteins of CDC6}

The present invention relates to a protein for inducing apoptosis, and more particularly, to a protein having a function of inducing apoptosis due to deletion of a part of the CDC6 protein and an apoptosis inducing agent containing the protein as an active ingredient.

Cell Survival-Death Reversible Controlling Technology is a next-generation key technology for developing apoptosis modulating therapy. Recent studies have shown that the causes of various diseases are attributable to abnormal functions of apoptosis signaling machinery (Nicholson, DW, Nature , 407, 810-816, 2000). Apoptosis therapy regulates the growth and death of abnormal cells by inducing apoptosis or inhibiting apoptosis. The purpose of apoptosis therapy is not only to block disease progression by abnormal cell death but also to function abnormal cells. By converting to cells of the underlying treatment of the disease.

Diseases applied to apoptosis control therapy that are competitively developed around the world are various senile and degenerative diseases such as cancer, dementia, Parkinson's disease, AIDS and aging including leukemia. However, as recent studies have shown that the onset of most diseases ultimately results from abnormal functioning of apoptosis signaling mechanisms, apoptosis therapy is the underlying technology that can be applied to the treatment of a wider range of diseases. have.

Apoptosis induction therapy can be used to treat diseases by blocking pathological growth of cells and inducing spontaneous apoptosis of pathological cells. Existing widespread necrosis pharmacotherapy has the side effect of killing pathologic cells and at the same time many cytotoxic enzymes (e.g. Lysozyme) are released to the normal cells around them. It becomes bigger. However, apoptosis induction therapy induces spontaneous killing of pathological cells, where many cytotoxic substances are mostly cleaved by caspase enzymes and lose their function during apoptosis. Is wrapped in an apoptotic body and undergoes a process of pagocytosis by macrophage. Therefore, it is known that cytotoxic substances do not leak to the outside and thus do not exhibit toxicity to surrounding cells.

Programmed Cell Death (APoptosis) is an active cell death process that requires energy and shows characteristic cell morphology. When apoptosis signal is transmitted, apoptosis is performed after determining apoptosis in the cell, and the biochemical and morphological changes of characteristic cells are shown in the execution phase. That is, the cells shrink and fall off from adjacent cells, and the cell membrane becomes bubble-like (blebbing), further condensing the nucleus and DNA in the nucleus into small oligonucleotide fragments. It is cut and forms an apoptotic body. The apoptotic body thus formed undergoes a series of processes in which pagocytosis is carried out by macrophages, resulting in cell death. Apoptosis is a complex intracellular process, but the intracellular determination of apoptosis is not easy, but once it is determined, several substeps are performed in order for apoptosis to function properly. Most morphological changes are caused by aspartic acid specific cysteine protease (caspase), an activating enzyme during cell death (Hengartner, MO, Nature , 407, 770-776). All caspases known to date are known to cut specific cleavage sites, namely, the Aspartate-X binding site of the substrate (Thornberry, NA, et. Al., J. Biol. Chem., 272, 17907- 17911, 1997). Large subunits of poly (ADP-ribose), polymerase (PARP) (Nicholson, DW, et al., Nature , 376, 37-43, 1995), replication factor C as substrates of caspase 3 (large subunit) (Rheaume, E., et al., EMBO J. , 16, 6346-6354, 1997), initiation factor 2 of eukaryotic cells (Marissen, WE, et al., J. Biol) Chem ., 275, 9314-9326, 2000), p21waf1 (Park, JA, et al., Eur. J. Biochem ., 257, 242-248, 1998) and Rb protein (Tan, XQ, et al., J. Biol. Chem ., 272, 9613-9616, 1997) and the like, many of these proteins are known for cell division and cell cycle. Thus, proteins that regulate cell cycles are thought to be important target molecules of caspase enzymes in apoptosis.

DNA replication in eukaryotic cells is tightly regulated and linked to cell division. DNA replication occurs during the S phase of the cell cycle, and replication precursors must be formed before replication can begin. The precursor of the replication complex is formed by binding the CDC6 protein and six MCM proteins to the replication origin of the chromosome, where the CDC6 protein is necessary for the MCM proteins to bind to the replication origin. DNA replication proceeds by phosphorylating the serine amino acid residues of the N-terminal portion of the CDC6 protein by the active cyclin A / cyclin-dependent kinase 2. Many reports indicate that phosphorylated CDC6 proteins block DNA replication by moving towards the cytoplasm in the cell nucleus (Jiang, W., et. Al.,Proc. Natl. Acad. Sci. USA, 96, 6193-6198, 2000; Peterson, B. O., et. Al.,EMBO J., 18, 396-410, 1999), and these CDC6 proteins are recognized as important factors for DNA replication and active research is being conducted.

Apoptosis and proliferation are opposing intracellular processes that maintain the homeostasis of multicellular organisms. Recently, a series of studies have reported several common regulators in the opposing system of apoptosis and cell proliferation. P53, p21, retinoblastoma protein, and protein dephosphatase, cdc25, are factors that regulate cell division. , C-Myc, a tumor protein, E2F-1, a transcriptional regulator, and E1A, a viral tumor protein, regulate cell death. In addition, recently, cyclin-dependent kinase 2, a key protein that regulates cell division, is required to be activated not only in cell division but also in apoptosis, and when cells selectively inhibit the activity of these enzymes, There has been a series of reports (Ying Hua Jin, et. Al., J. Biol. Chem ., 276, 30256-30263, 2000) that not only blocks the progress of death but also restores cell growth to normal levels. These reports suggest that cyclin dependent kinase 2 plays an essential role in the phase of cell growth-killing, and the point of action of the enzyme is a reversible determining step of cell growth-killing. However, there have been no reports of cyclin-dependent kinase 2 substrates directly related to apoptosis and apoptosis inhibitors using these proteins.

Therefore, the present inventors endeavored to develop apoptosis inducer that can be applied to various cancer diseases including leukemia by controlling cell growth and death. As a result, a protein which removed some amino acid sequences of CDC6 protein, which plays an important role in cell replication, is apoptosis process. The present invention was completed by finding that the protein has an excellent effect of inducing apoptosis and revealing that the protein can be used as an apoptosis inducing agent.

It is an object of the present invention to provide a protein in which some amino acids of the CDC6 protein inducing apoptosis are deleted and an apoptosis inducing agent containing the protein as an active ingredient.

FIG. 1 is a photograph showing that PARP and CDC6 proteins are commonly cut during various spontaneous apoptosis processes.

A: Ginsenoside Rh2 (G-Rh2) treatment, B: TRAIL treatment,

C: Etoposide treatment, D: Taxol treatment

FIG. 2 is a photograph showing that CDC6 protein is blocked by DEVD, a selective inhibitor of caspase 3, an effector caspase during spontaneous apoptosis.

a: DEVD-fmk and / or ginsenoside Rh2 (G-Rh2) treatment,

b: DEVD-cho, VEID-cho or IETD-cho, and etoposide, taxol or ginsenoside Rh2 (G-Rh2) treatment,

c: caspase 3, 6, 7, 8, or 9 and / or DEVD-cho treatment

Figure 3 is a photograph showing that the D442N variant protein modified with amino acid 442 of the normal CDC6 protein is not cleaved by caspase 3.

A: sequence comparison of normal CDC6 protein and variant proteins,

B: caspase 3 treatment to normal CDC6 protein and variant proteins,

C: Caspase 3, etoposide, taxol or ginsenoside Rh2 (G-Rh2) treatment of normal CDC6 protein and D442N variant protein

Figure 4 shows that CDC6 protein is cleaved at the SEVD ₄₄₂ -G binding site by caspase 3 during the apoptosis process.

A: comparison of CDC6 protein cleavage site sequence in humans and mice,

B: Schematic diagram of functional site of CDC6 protein

Figure 5a is a photograph showing that the induction of apoptosis in terms of cell membrane blebbing (bleeding) when induction of apoptosis after overexpressing the CDC6 (1-442) protein of the present invention in human uterine cancer cell line.

FIG. 5B graphically illustrates the results of FIG. 5A.

Figure 6a shows that the induction of apoptosis through FACS (Fluorescence Activated Cell Sorter) analysis when overexpressing the CDC6 (1-442) protein of the present invention in human uterine cancer cell line.

FIG. 6B graphically illustrates the results of FIG. 6A.

7 is a graph showing the membrane bleeding phenomenon with time after overexpressing the CDC6 (1-442) protein of the present invention in human uterine cancer cell lines.

8 is a photograph showing the DNA condensation and fragmentation by overexpressing the CDC6 (1-442) protein of the present invention in human uterine cancer cell lines and staining the nucleus.

9 is a graph measuring the activity of caspase 3 over time after overexpressing the CDC6 (1-442) protein of the present invention in human uterine cancer cell lines.

Figure 10a shows the results of FACS analysis after overexpressing the CDC6 (1-442) protein of the present invention in human uterine cancer cell line.

FIG. 10B graphically illustrates the FACS results of FIG. 10A.

Figure 11a shows a human uterine cancer cell line containing the CDC6 (1-442) protein, CDC6 (1-404) protein, CDC6 (1-324) protein, CDC6 (1-300) protein and CDC6 (1-204) protein of the present invention. The cell membrane bleeding phenomenon after overexpression.

Figure 11B shows a human uterine cancer cell line containing the CDC6 (1-442) protein, CDC6 (1-404) protein, CDC6 (1-324) protein, CDC6 (1-300) protein and CDC6 (1-204) protein of the present invention. It is a graph showing the membrane bleeding phenomenon at 24 hours or 36 hours after overexpression.

In order to achieve the above object, the present invention provides an apoptosis inducing protein comprising amino acid sequence 1 to 179 of the CDC6 protein described in SEQ ID NO: 1 , the amino acid sequence 462 to 488 is deleted.

The present invention also provides an apoptosis inducer containing the protein as an active ingredient.

Hereinafter, the present invention will be described in detail.

The apoptosis inducing protein of the present invention comprises amino acid sequence 1 to 179 that can interact with cyclin A or c-Myc protein, etc. among the CDC6 protein described in SEQ ID NO: 1 , nuclear emission signal for moving the protein into the cytoplasm (Nuclear Export Signal; NES) characterized in that the amino acid sequence of Nos. 462 to 488 is deleted.

The apoptosis inducing protein comprises amino acid sequence 1 to 179 of the CDC6 protein described in SEQ ID NO: 1 and deletion of amino acid sequence 462 to 488, in addition to part or all of the amino acid sequence of 180 to 442 It is preferably a protein comprising, amino acid sequence 1 to 442 of the CDC6 protein described in SEQ ID NO: 2 , amino acid sequence 1 to 404 of the CDC6 protein described in SEQ ID NO: 3 , CDC6 protein described in SEQ ID NO: 4 More preferably, it is a protein consisting of amino acid sequences 1 to 324, amino acids 1 to 300 of the CDC6 protein of SEQ ID NO: 5 or amino acids 1 to 204 of the CDC6 protein of SEQ ID NO: 6 .

CDC6 protein is cleaved during the apoptosis process, specifically, caspase 3 is cleaved at the aspartic acid site 442 so that fragments containing amino acid sequences 1 to 442 induce apoptosis.

Nuclear Localization Sequence (NLS) exists between amino acids 56-95 of the CDC6 protein ( FEBS letters , 447, 292-206; Lauric, MD, et. Al., The Journal of Biological Chemistry , 276, 26947-26954, 2001). The sequence is a domain that transfers the CDC6 protein into the cell nucleus to participate in DNA replication. Overexpressing the CDC6 (111-560) protein in which the domain region was removed in the cell shows that the CDC6 (111-560) protein only spreads throughout the cell but does not selectively enter the nucleus, thus exhibiting an effect on apoptosis. Do not. On the other hand, SEQ ID NO: 2 (1-442), SEQ ID NO: 3 (1-404), SEQ ID NO: 4 (1-324), SEQ ID NO: 5 (1-300) or SEQ ID NO: 6 (1) including the nuclear position sequence In the case of the protein consisting of -204), it moves into the cell nucleus, resulting in an apoptosis effect. However, the nuclear position sequence alone does not induce cell death. For example, SEQ ID NO: 1 in the case of CDC6 CDC6 protein or SEQ ID NO: 1 (1-100) protein consisting of one to 100 amino acid sequence in Figure 1 does not lead to but including the nuclear location sequence apoptosis (Fig. 7 and FIG. 11 ). That is, the nuclear position sequence is a domain necessary for inducing apoptosis inducing proteins of the present invention, but does not induce apoptosis with the domain alone.

The CDC6 protein is present as a factor that forms a replication complex precursor when DNA replication begins, and is phosphorylated by activated cyclin A / cyclin-dependent kinase 2 to migrate out of the nucleus when DNA replication begins. This transfer of CDC6 protein into the cytoplasm is intended to maintain intracellular homeostasis to prevent DNA replication in the nucleus. Nuclear Export Signals (NES) for migration to the cytoplasm have been reported to exist at amino acid sequences 462-488 for CDC6 proteins (Lauric, MD, et. Al., The Journal of Biological Chemistry , 276, 26947-26954, 2001). If the CDC6 protein is present in the nucleus and causes intracellular damage, normal cells will undergo a process of release or degradation of the CDC6 protein into the cytoplasm by the cellular homeostasis system. On the other hand, since all of the apoptosis-inducing proteins of the present invention do not have a nuclear release signal, spontaneous killing of cells is induced because the mutant CDC6 proteins are not transferred to the cytoplasm even if the cells are damaged in the nucleus.

In addition, it has been reported that amino acid sequences 1 to 179 of the CDC6 protein have a sequence that interacts with Orc1 protein, cdc18 protein, cyclin A protein or c-Myc protein (Weinreich, M., et al.,Proc. Natl. Acad. Sci. USA, 98, 11211-11217, 2001; Takayama, M., et al.,FEBS Letters, 477, 43-48, 2000; Saha, P., et al.,Mol. Cell. Biol., 18 (5), 2758-2767, 1998; Williams, R. S., et al.,Proc. Natl. Acad. Sci. USA, 94, 142-147, 1997). Orc1 and cdc18 proteins are involved in DNA replication and play an important role in DNA replication rather than apoptosis. On the other hand, cyclin A protein is known to increase the activity of the kinase by binding to cyclin-dependent kinase 2 not only in cell division but also in apoptosis process, and c-Myc protein is also a lower level protein in cell proliferation and death process. It is known to activate. Therefore, it is expected that the apoptosis inducing protein of the present invention interacts with cyclin A or c-Myc, inducing cell growth and apoptosis.

In the normal cell group treated with etoposide, an apoptosis inducing agent, about 20% of apoptosis was induced, whereas in the cell group treated with etoposide to about 80% of cells, the cell group overexpressed the protein of the present invention. Death is induced (see FIG. 5 ). In addition, as a result of observing the number of apoptosis cells (subG1 cell number) through FACS (Fluorescence Activated Cell Sorter) analysis, less than 10% of the total cells in the sub-G1 phase were treated with etoposide. On the other hand, when etoposide was treated to cells overexpressing the protein of the present invention, the number of cells in the sub-G1 stage was significantly increased to 15% or more (see FIG. 6 ). That is, when the apoptosis inducing protein of the present invention is treated with an apoptosis inducing agent, it can be seen that apoptosis is significantly increased.

In addition, overexpression of the protein of the present invention without giving apoptosis conditions to normal cells results in bleeding in the cell membrane (see FIG. 7 ), and DNA condensation and fragmentation in the cell nucleus. 8 ). In addition, the caspase 3 activity, which is activated during apoptosis, is increased by 2 to 3 times (see FIG. 9 ). Therefore, it can be seen that apoptosis is induced even when the protein of the present invention is treated alone.

The present invention also provides an apoptosis inducer containing the protein as an active ingredient.

The apoptosis inducer of the present invention comprises the amino acid sequence of amino acids 1 to 179 of the CDC6 protein set forth in SEQ ID NO: 1 , and contains a protein from which amino acid sequences 462 to 488 are deleted. The protein comprises amino acid sequences 1 to 179 of the CDC6 protein as set forth in SEQ ID NO: 1 and deletions of amino acid sequences 462 to 488, and further includes a part or all of amino acid sequences 180 to 442. Preferably, the amino acid sequence 1 to 442 of the CDC6 protein described in SEQ ID NO: 2 , the amino acid sequence 1 to 404 of the CDC6 protein described in SEQ ID NO: 3, and the CDC6 protein described in SEQ ID NO: 4 More preferably, it is a protein consisting of amino acid sequence No. 324, amino acid sequence No. 1 to 300 of the CDC6 protein described in SEQ ID NO: 5 or amino acid sequence no . 1 to 204 of the CDC6 protein described in SEQ ID NO: 6 .

In addition, the apoptosis inducer of the present invention may contain one or more kinds of proteins selected from the group consisting of proteins represented by SEQ ID NO: 2 to SEQ ID NO: 6 as an active ingredient.

The apoptosis inducing agent is breast cancer (Parton, M., et al., British Medical Journal, 322, 1528-1532, 2001), rectal cancer (Rampino, N., et al.,Science, 275,967-969, 1997), thyroid cancer (Lin, J.D., British Medical Journal, 322, 1525-1527, 2001) and various cancers and rheumatoid arthritis (Perlman, H., et al.,Curr. Mol. Med., 5, 597-608, 2001).

The apoptosis inducing agent of the present invention may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, granules, suspensions, emulsions, It can be formulated into syrups, other liquids.

In particular, the apoptosis inducing agents of the present invention may be formulated for oral administration, for example tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or It is formulated with elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above.

In addition, the apoptosis inducer of the present invention can be administered parenterally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. To formulate into a parenteral formulation, the apoptosis-inducing protein is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

The dosage of the active ingredient according to the present invention is appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, and the severity of the disease to be treated, but generally in adults one day The apoptotic protein may be administered once to several times in an amount of 0.1 to 100 mg per kg of body weight, preferably 1 to 10 mg.

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

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Cleavage of CDC6 Protein in Induction of Apoptosis

The present inventors conducted the following experiment to observe the change of CDC6 protein when induction of apoptosis by various apoptosis inducers.

Ginsenoside Rh2 (G-Rh2), Etoposide, Taxol, or Tumor necrosis factor (TNF) -related Apoptosis Inducing Ligand (TRN), apoptosis inducers After treatment in hepatocellular carcinoma cell line was observed the change of cells over time. Etoposide, a topoisomerase II inhibitor, is known as a drug that blocks the cell cycle at S phase of the cell cycle. Taxol stabilizes the assembly of microtubules to stabilize the cell division process. It is known to induce apoptosis based on this mechanism of action as a drug to block. TRAIL is known as a protein that induces apoptosis via death receptors (DR4, DR5), and apoptosis by TRAIL is caused by apoptosis via receptors, unlike apoptosis via mitochondria. It is known to proceed. Ginsenoside Rh2 is known for its apoptosis mechanism via mitochondria and shows a typical cell morphology of apoptosis.

Specifically, a 100 mm culture vessel for human uterine cancer cell line (HeLa cell) (Seoul National University College of Medicine Cancer Research Institute) and liver cancer cell line (SK-HEP-1) (Seoul National University Medical University Cancer Research Institute) with a cell count of 1x10 ⁶ . After incubation for 24 hours at 37 ℃, carbon dioxide 5% conditions to provide an environment in which cells can grow, each drug was treated to the cells and cells were collected over time. 85 μM etoposide, 80 nM taxol or 12 μM ginsenoside Rh2 drug was treated in human uterine cancer cell lines and 43 μM TRAIL in human liver cancer cell lines. TRAIL uses a hepatic cancer cell line because apoptosis occurs because of receptor-mediated apoptosis without associated receptors. 12 μM ginsenoside Rh2 induced apoptosis in serum-free conditions because apoptosis did not occur in the presence of serum, and other drugs induced apoptosis in the presence of 5% calf serum. The collected cells were ground with a homogenizer, and only nuclear fractions were collected separately to dissolve the nuclear membrane with a lysis buffer. The protein obtained above was quantified, and the same amount of protein was dissociated in an SDS-PAGE gel, followed by an antibody immunoassay using an anti-CDC6 antibody and an anti-PARP antibody. PARP is a typical substrate of caspase 3 and the cleavage of PARP is used as a measure of induction of apoptosis.

As a result, a cleaved CDC6 protein of about 49 kDa size was observed in common in each cell death condition ( FIG. 1 ). Such cleavage of the CDC6 protein was common in other apoptosis induction systems.

Example 2 Cleavage of Selective CDC6 Protein by Caspase 3

<2-1> Treatment of Caspase 3 Inhibitors on Human Uterine Cancer Cell Lines

Caspase 3 enzyme, a key enzyme in the apoptosis process, selectively cleaves Asp-X binding sites, and the present inventors have found that caspase 3 is a caspase 3 selectively cleaved during apoptosis. The experiment was performed by treating a third selective inhibitor, z-DEVD-Fmk (carbobenzoxy-Asp-Glu-Val-Asp-fluoromethylketone).

Specifically, human uterine cancer cell lines were placed in a 60 mm culture vessel with 3 × 10 ⁵ water and then cultured at 37 ° C. and 5% carbon dioxide conditions for 24 hours. After 24 hours, cells were collected after 3 hours with simultaneous treatment of 9.6 μM ginsenoside Rh2 and 100 μM z-DEVD-Fmk or only 9.6 μM ginsenoside Rh2. After collecting only the nuclear fractions of the collected cells and dissolving the nuclear membrane with a lysis buffer, proteins were quantified and dissociated in the SDS-PAGE gel by the same amount of immunoblot using anti-CDC6 antibody and anti-PARP antibody.

As a result, it was confirmed that CDC6 protein was cleaved in cells treated with ginsenoside Rh2 only, but the cleavage of CDC6 protein was suppressed in the experimental group treated with the caspase 3 inhibitor z-DEVD-Fmk ( FIG . 2A ). . The results showed that the CDC6 protein was cleaved by caspase 3 during the apoptosis process.

<2-2> In vitro of CDC6 protein in vitro A) cutting analysis

In order to determine the direct effect of caspase 3 on CDC6 protein, the present inventors examined the direct interaction of CDC6 protein with caspase 3 outside of the cell rather than in an intracellular experiment in which various factors exist. Specifically, cdc6 cDNA was subcloned and inserted into pCITE vector (Novagen). Using the forward primer (5'-AAGGATCCCTCAAACCCGATCCCAG-3 ') and SEQ ID NO: 8 reverse primer (5'-AAGGATCCTTAAGGCAATCCAG-3 described in') described in SEQ ID NO: 7 was performed PCR. 1 μl of each primer (25 pmol / μl) was added, and 1 μl of PCR Taq polymerase (Takara, Japan), 10 μl of reaction buffer (10 ×), and 8 μl of 2.5 mM dNTP were used as template DNA. After mixing with cdc6 cDNA, distilled water was added to make a total of 100 μl, and then turned once at 94 ° C. for 4 minutes, and then turned 30 times under conditions of 95 ° C. 30 seconds, 55 ° C. 2 minutes, and 72 ° C. 2 minutes. In the last cycle, PCR was performed under conditions of 1 minute at 94 ° C, 2 minutes at 55 ° C, and 5 minutes at 72 ° C. The product obtained by PCR and the pCITE vector were cleaved with BamHI, and then subjected to a ligation reaction at 16 ° C. for 4 hours using T4 DNA ligase (Takara, Japan) for subcloning.

In vitro transcription and translation experiments were performed using the vectors prepared above. In vitro cleavage assay was performed with the [ ³⁵ S] -S-Tag-CDC6 protein produced in this experiment. Cell extracts treated with 12 μM ginsenoside Rh2, etoposide or taxol were used. It was used as caspase 3 (can be used because the caspase 3 is active in the cell extract undergoing apoptosis). In addition, 100 μM Ac-DEVD-cho (N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde); selective inhibitor of caspase 3) or 100 μM Ac-VEID-cho (N-acetyl-Val-Glu -Ile-Asp-CHO (aldehyde); selective inhibitor of caspase 6) or 100 μM Ac-IETD-cho (N-acetyl-Ile-Glu-Thr-Asp-CHO (aldehyde); selective inhibitor of caspase 8) After treatment with each of the reaction at 37 ℃, dissociation in SDS-PAGE, a band of [ ³⁵ S] -S-Tag-CDC6 protein was detected by radiograph.

As a result, the [ ³⁵ S] -S-Tag-CDC6 protein was cleaved by ginsenoside Rh2, etoposide or Taxol treated apoptosis extract, and in the experimental group treated with the caspase 3 inhibitor, the [ ³⁵ S]- Cleavage of the S-Tag-CDC6 protein was inhibited. On the other hand, in the experimental group treated with Ac-VEID-cho, a selective inhibitor of caspase 6, or Ac-IETD-cho, a selective inhibitor of caspase 8, [ ³⁵ S] -S-Tag-CDC6 protein was cleaved. Therefore, it was confirmed that the cleavage of the [ ³⁵ S] -S-Tag-CDC6 protein was a selective cleavage by caspase 3 ( FIG . 2B ).

Next, in vitro cleavage analysis was performed using recombinant caspases on the [ ³⁵ S] -S-Tag-CDC6 protein to determine the more direct effect by caspase 3. Recombinant caspase 3, 6, 7, 8, or 9 and recombinant caspase 3 or 7 and inhibitors thereof [ ³⁵ S] -S-Tag-CDC6 protein (caspases 3 and 7 cleave the same conservative aspartic acid site) In vitro cleavage analysis was performed.

As a result, caspases 3 and 7 cleaved the [ ³⁵ S] -S-Tag-CDC6 protein, but when the inhibitor of caspase 3 was administered, the cleavage was suppressed. In addition, caspase 6, 8 or 9 did not cleave the [ ³⁵ S] -S-Tag-CDC6 protein. From the above results, it was confirmed once again that the CDC6 protein was selectively cleaved by caspase 3 like protein cleavage enzyme (caspase 3-like protease: caspase 3 or caspase 7) ( FIG . 2C ).

Example 3 Cleavage Site Mapping of CDC6 Protein by Caspase 3

In order to identify the cleavage site of CDC6 protein by caspase 3, the present inventors substituted aspartic acid with asparagine around 49 kDa from the N terminus of CDC6 protein by performing point mutations. In this case, the primers set forth in SEQ ID NO: 9 for D404N, the primers set forth in SEQ ID NO: 10 for D442N, and the primers set forth in SEQ ID NO: 11 for D502N were tested. Confirmed. In vitro cleavage analysis was performed with the mutant protein ( A of FIG. 3 ) obtained by in vitro transcription and translation analysis with the mutated DNA obtained by substitution.

As a result, when each mutant protein was reacted in the presence of recombinant caspase 3, the [ ³⁵ S] -S-Tag-CDC6 proteins of D404N and D502N were cleaved by caspase 3, but [ ³⁵ S]-of D442N S-Tag-CDC6 protein was not cleaved by caspase-3 (B in Fig. 3). In addition, it was confirmed that the D442N mutant protein was not cleaved not only by recombinant caspase 3 but also by etoposide, taxol, or ginsenoside Rh2 treated apoptosis extract ( FIG . 3C ). Thus, the cutting of the CDC6 protein by caspase-3 was found to occur at time 442 aspartic these caspase-3 cleavage site was found that the amino acid sequences stored in other mammals, the mouse (A of FIG. 4).

Example 4 Preparation of Expression Vector Expressing CDC6 Protein Segment

The inventors prepared an expression vector expressing a CDC6 protein fragment in order to determine the function of the CDC6 protein cleaved during apoptosis. Specifically, when the DNA corresponding to the CDC6 protein fragment is overexpressed in a cell, a pCS2 + GFP vector tagged with green fluorescence (tagging) to recognize the overexpressed cell (received from Dr. Kwang-yeol Lee of Harvard University) Subcloning). In the case of cDNA corresponding to the wild type CDC6 protein, a forward primer as shown in SEQ ID NO: 12 and a reverse primer as shown in SEQ ID NO: 13 were used, and CDC6 having a amino acid sequence of 1 to 442 amino acids of CDC6 protein (1-442). ) CDNA corresponding to the protein was used as the forward primer described in SEQ ID NO: 14 and the reverse primer described in SEQ ID NO: 15 , corresponding to the CDC6 (111-560) protein having a 111 to 560 amino acid sequence of CDC6 protein In the case of cDNA, amplification was carried out by PCR using a forward primer of SEQ ID NO: 16 and a reverse primer of SEQ ID NO: 17 and then inserted into the XhoI and StuI restriction enzyme sites of the pCS2 + GFP vector. 1 μl of each primer (25 pmol / μl) was added, and 1 μl of PCR Taq polymerase (Takara, Japan), 10 μl of reaction buffer (10 ×), and 8 μl of 2.5 mM dNTP were used as template DNA. After mixing with cdc6 cDNA, distilled water was added to make a total of 100 μl, and then turned once at 94 ° C. for 4 minutes, and then turned 30 times under conditions of 95 ° C. 30 seconds, 55 ° C. 2 minutes, and 72 ° C. 2 minutes. In the last cycle, PCR was performed under conditions of 1 minute at 94 ° C, 2 minutes at 55 ° C, and 5 minutes at 72 ° C. The product obtained by PCR and the pCS2 + GFP vector were cleaved with XhoI and StuI, and then subjected to a ligation reaction at 16 ° C. for 4 hours using T4 DNA ligase (Takara, Japan). Subcloning.

Example 5 Apoptosis Analysis by CDC6 (1-442) Protein in Apoptosis Conditions

<5-1> Analysis of Cell Membrane upon Induction of Apoptosis in Cells Overexpressing CDC6 (1-442) Protein

Based on blebbing in the cell membrane, a typical cellular change in the process of apoptosis, the cell membrane bleeding was counted as a percentage of the cells overexpressing the CDC6 (1-442) protein (overexpressed cells). Heavy cell membrane bleeding occurs / cell number overexpressed x 100).

Specifically, human cervical cancer cells were placed in a 35 mm culture vessel with 1.5 × 10 ⁵ water and then 24 hours later using polyFect, pCS2 + GFP (control), pCS2 + GFP-cdc6, pCS2 + GFP-cdc6 (1-442), pCS2 + GFP-cdc6 (111-560) was transfected into cells to overexpress each protein. At 12 hours after the overexpression experiment, 85 μM of etoposide was treated. At 12 hours after the etoposide treatment, the morphology of the cells was photographed, and the number of cell membrane bleeding cells was counted among the overexpressed cells. As a control, only the original vector tagged with green fluorescence was overexpressed.

As a result, up to 80% of cell membrane bleeding in cells overexpressing CDC6 (1-442) protein was observed in etoposide compared to control, CDC6 protein or cell group overexpressing CDC6 (111-560) protein. It can be seen that significantly increased the apoptosis induction action ( Fig. 5a and 5b ).

<5-2> Analysis of Cell Cycle Change in Induction of Apoptosis in Cells Overexpressing CDC6 (1-442) Protein

When apoptosis occurs, the ratio of the subG1 phase (subG1 phase) of the cell cycle is increased, and the degree of apoptosis induction by CDC6 (1-442) protein was measured based on the evaluation criteria.

Specifically, human cervical cancer cells were placed in a 35 mm culture vessel with 1.5 × 10 ⁵ water and then 24 hours later using polyFect, pCS2 + GFP (control), pCS2 + GFP-cdc6, pCS2 + GFP-cdc6 (1-442), pCS2 + GFP-cdc6 (111-560) were infected with cells to overexpress each protein. At 12 hours after the overexpression experiment, 85 μM of etoposide was treated, and at 12 hours after etoposide treatment, the cells were trypsinized to remove the cells from the container and collected and fixed with 75% ethanol. It was stained with DAPI staining solution and analyzed by FACS analyzer.

As a result, up to 17.6% of the sub-G1 levels in the cell group overexpressed the CDC6 (1-442) protein, which is etoposide compared to the control group, the CDC6 protein or the cell group overexpressed the CDC6 (111-560) protein. It can be seen once again that significantly increases apoptosis-inducing action by ( FIG. 6A and FIG. 6B ).

Example 6 Analysis of Induction of Apoptosis by CDC6 (1-442) Protein Only

The present inventors overexpressed the CDC6 (1-442) protein to induce apoptosis with its own action, and in time, after overexpressing each DNA in human uterine cancer cell lines in the same manner as in Example 5 In order to find out the change of the cell according to the following experiment was carried out.

<6-1> Cell membrane change analysis

Typical cell changes during apoptosis were shown by counting the number of cell membrane bleeding cells among cells overexpressing the CDC6 (1-442) protein using the bleeding phenomenon in the cell membrane. Human uterine cancer cells were placed in a 35 mm culture vessel with 1.5 × 10 ⁵ water and incubated at 37 ° C. and 5% carbon dioxide for 24 hours, followed by pCS2 + GFP (control), pCS2 + GFP− using PolyFect. cdc6, pCS2 + GFP-cdc6 (1-442) and pCS2 + GFP-cdc6 (111-560) were infected with cells to overexpress each protein. From this, the shape of the cells at 24, 36, and 48 hours was photographed, and the number of cells in which the bleeding phenomenon was observed in the cell membrane among the overexpressed cells was counted and expressed as a percentage.

As a result, there was no significant change in the control group, CDC6 protein or CDC6 (111-560) protein until 48 hours after cell infection, but in the experimental group overexpressed with CDC6 (1-442) protein, 18.1% at 24 hours and 66.3 at 36 hours. %, 48 hours at 70.9% of the overexpressed cell membrane bleeding phenomenon was found to increase, indicating that the apoptosis increases with time ( Table 1 , Figure 7 ).

Membrane bleeding induction rate according to overexpression of CDC6 protein in human uterine cancer cell line (mean value ± standard deviation%) 24 hours 36 hours 48 hours Control 2.86 ± 2.05 3.18 ± 1.29 7.56 ± 2.51 CDC6 Protein 3.12 ± 0.36 9.82 ± 3.85 12.04 ± 2.35 CDC6 (1-442) Protein 18.16 ± 0.93 66.37 ± 4.29 70.98 ± 6.09 CDC6 (111-560) Protein 2.86 ± 1.14 7.33 ± 1.40 14.87 ± 0.34

<6-2> Analysis of Condensation and Fragmentation in Nuclei

In order to observe nuclear condensation and fragmentation by DAPI staining, which is another indicator of apoptosis, PBS (Phosphate Buffer Saline) (PBS) was observed at 36 hours after overexpressing CDC6 proteins in the same manner as in Example 6-1. After washing with 4% paraformaldehyde was fixed. After 300 μl of DAPI stain was added to the fixed cells, the reaction was performed at 37 ° C. for about 10 minutes, and then washed twice with PBS. Then, a drop of 50% glycerol was added, covered with cover glass, and observed under a fluorescence microscope. Photographed.

As a result, there was no change in the nucleus in the control group, the cell group overexpressing the CDC6 protein or the CDC6 (111-560) protein, whereas in the experimental group overexpressing the CDC6 (1-442) protein, typical nuclear condensation and fragmentation were observed ( 8 ).

<6-3> Analysis of increased activity of caspase 3

Since the increased activity of caspase 3, an effector caspase in the process of apoptosis, is a measure of apoptosis. After overexpressing CDC6 proteins in the same manner as in Example 6-1, cells were collected at 36 hours, dissolved in an IP lysis buffer, and extracted with intracellular proteins, followed by experiments with the same amount of protein. Caspase 3 activity was determined by measuring fluorescence using a substrate that fluoresces when caspase 3 is cleaved. N-acetyl-Asp-Glu-Val-Asp-AFC (7-amino-4-trifluoromethyl-coumarin) at 50 μg and 200 nM of each cell extract protein; Caspase 3 fluorescent substrate; Caspa This property is used to measure caspase activity because it does not emit light before cleavage with a third fluorescent substrate but emits fluorescence when cleaved by caspase. The reaction buffer (20 mM Hepes, pH 7.4), 100 mM NaCl , 10 mM DTT, 0.1% CHAPS, 10% sucrose) was reacted for 1 hour at 37 ℃ and the fluorescence was measured at an emission wavelength of 535 nm (excitation wavelength 405 nm).

As a result, caspase 3 activity per unit protein mass of the cell extract overexpressing the CDC6 (1-442) protein was 3.5 at 36 hours after cell infection compared to the control, CDC6 protein or the cell group overexpressing the CDC6 (111-560) protein. It was shown to increase up to fold ( FIG. 9 ).

<6-4> Cell Cycle Change Analysis

When apoptosis occurs, the ratio of the sub-G1 phase in the cell cycle is increased, and the degree of apoptosis caused by CDC6 (1-442) protein was measured based on the evaluation criteria. Specifically, CDC6 proteins were overexpressed in the same manner as in Example 6-1, after treatment with trypsin for 36 hours after cell infection, the cells were detached and collected and fixed in 75% ethanol. This was stained with DAPI staining solution and analyzed by FACS analyzer.

As a result, up to 20% of the sub-G1 levels in the cell group overexpressing the CDC6 (1-442) protein were significantly increased induction of apoptosis compared with the control (mock) or cell group overexpressing the CDC6 protein. It can be seen that once again ( Figs. 10a and 10b ).

Example 7 Identification of Apoptosis Inducing Domain by Deletion Protein

<7-1> Preparation of vector expressing deletion protein

The present inventors prepared a deletion mutant in 100 amino acid units in order to determine which domain of the CDC6 (1-442) protein to induce apoptosis.

Specifically, ORC (Origin Replication Complex) homologous domain box I is present in amino acid sequence 170-204, box III is amino acid sequence 273-294, box IV is amino acid sequence 309-325, box V is amino acid sequence 347 -360, box VI is located at amino acid sequence 371-402, amino acid sequence 1-100 with phosphorylation site by cyclin dependent kinase, amino acid sequence 1-204 in the range including box I, box III A CDC6 deletion mutation was made in amino acid sequence 1-300 comprising amino acid sequence, amino acid sequence 1-324 comprising box IV and amino acid sequence 1-404 comprising all ORC homologous domain boxes. Each forward primer commonly used the primer set forth in SEQ ID NO: 16, and the reverse primer of DNA corresponding to the CDC6 (1-100) protein corresponds to the primer set forth in SEQ ID NO: 19 , the CDC6 (1-204) protein. The reverse primer of the DNA is the primer of SEQ ID NO: 20 , the reverse primer of the DNA corresponding to the CDC6 (1-300) protein, the reverse primer of the DNA of SEQ ID NO: 21 , the DNA of the CDC6 (1-324) protein reverse primer in the amplification by the reverse primer of the DNA for the primer and CDC6 (1-404) protein described in SEQ ID NO: 22 using primers described in SEQ ID NO: 23 respectively, the top cdc6 cDNA by PCR using as a template And inserted into the XhoI and StuI restriction enzyme sites of the pCS2 + GFP vector, respectively.

<7-2> Apoptosis Effect Analysis

The present inventors analyzed the effect on the apoptosis of the deleted protein using the expression vector prepared in Example <7-1>.

Specifically, CDC6 (1-442) protein, CDC6 (1-404) protein, CDC6 (1-324) protein, CDC6 (1- 300) The number of cells in which cell membrane bleeding occurred among cells overexpressing the protein or the CDC6 (1-204) protein was shown by counting. Human uterine cancer cell lines were placed in a 35 mm culture vessel with ^1.5 × 10 ⁵ water and then 24 hours later using polyfects, pCS2 + GFP (control), pCS2 + GFP-cdc6, pCS2 + GFP-cdc6 (1-442), pCS2 + GFP-cdc6 (1-404), pCS2 + GFP-cdc6 (1-324), pCS2 + GFP-cdc6 (1-300), pCS2 + GFP-cdc6 (1-204) or pCS2 + GFP-cdc6 (1 -100) were infected with the cells to overexpress each protein. From this, the shape of the cells at 24 or 36 hours was photographed, and the number of cells in which the bleeding phenomenon was observed in the cell membrane among the overexpressed cells was measured.

As a result, about 36% of cells overexpressed CDC6 (1-442) protein at 36 hours after infection, 78% of CDC6 (1-404) protein and 78% of CDC6 (1-1-) protein. 324) showed 74% for membrane, 79% for CDC6 (1-300) protein, and 58% for CDC6 (1-204) protein. On the other hand, CDC6 (1-100) protein did not show a significant effect. Therefore, the smallest unit inducing apoptosis was observed with the CDC6 (1-204) protein ( FIGS. 11A and 11B ).

<Application Example 1> Cancer

When cells are not normally differentiated or damaged by external stimuli, cells choose programmed cell death (apoptosis) to maintain homeostasis. However, it is known that cancer may survive cancer cells while maintaining a balance between proliferation and spontaneous death by abnormal modification of these genes, and at the same time, defects in the spontaneous cell death system are involved. Mutations in the spontaneous cell death system may also be resistant to chemotherapeutic drugs or radiation treatments used to treat cancer. In order to effectively treat cancer, it has been discussed that cells damaged by a system that induces spontaneous cell death induce spontaneous cell death or enhance response to therapeutic drugs (Thompson, C.B.,Science, 267, 1456-1462, 1995; Sjostrom, J., et. al.,British Medical Journal, 322, 1538-1539, 2001). In the case of rectal cancer, the function of bax protein, a pro-apoptotic protein that promotes spontaneous cell death, is known to be lost, which suggests that bax plays an important role in the development of rectal cancer (Rampino, N., et. al.,Science, 275, 967-969, 1997). It has also been reported in preclinical trials that apoptosis by bax is sensitive to chemotherapy or radioactivity (Reed, J.C.,Sem. Hematol., 34, 9-19, 1997). Therefore, proteins which are part of the CDC6 protein according to the present invention showing the inducing effect of spontaneous cell death can be used to treat various cancers.

<Application Example 2> Rheumatoid Arthritis

Rheumatoid arthritis is a disease caused by decreased cell death along with increased cell proliferation and cell migration of synovial lining. Abnormal apoptosis and increased cell proliferation of synoviocytes similar to fibroblasts or macrophages lead to synovial hyperplasia, which causes cartilage and bone destruction. Recently, there are reports that many substances that control apoptosis and proliferation play an important role in the treatment of rheumatoid arthritis (Perlamn, H., et. Al., Curr. Mol. Med., 5, 597-608, 2001 ). Thus, proteins that have been deleted from a portion of the CDC6 protein according to the present invention showing the inducing effect of spontaneous apoptosis can be used to treat rheumatoid arthritis.

As described above, the CDC6 (1-404) protein, the CDC6 (1-300) protein and the CDC (1-204) protein of the present invention is a substance that induces apoptosis under normal cell growth conditions and regulates cell death By blocking the progression of the disease due to the pathological growth of the cell through can be usefully used to treat the disease. In particular, it can be effective in combating disease by inhibiting abnormal proliferation and killing of cancer cells through apoptosis induction therapy to various cancer diseases in which cells are not normally differentiated and continue to proliferate ideally.

<110> LEE, Seung Ki <120> Apoptotic inducer containing deleted proteins of CDC6 <130> 2p-07-05 <160> 23 <170> KopatentIn 1.71 <210> 1 <211> 560 <212> PRT <213> Homo sapiens <400> 1 Met Pro Gln Thr Arg Ser Gln Ala Gln Ala Thr Ile Ser Phe Pro Lys   1 5 10 15 Arg Lys Leu Ser Arg Ala Leu Asn Lys Ala Lys Asn Ser Ser Asp Ala              20 25 30 Lys Leu Glu Pro Thr Asn Val Gln Thr Val Thr Cys Ser Pro Arg Val          35 40 45 Lys Ala Leu Pro Leu Ser Pro Arg Lys Arg Leu Gly Asp Asp Asn Leu      50 55 60 Cys Asn Thr Pro His Leu Pro Pro Cys Ser Pro Pro Lys Gln Gly Lys  65 70 75 80 Lys Glu Asn Gly Pro Pro His Ser His Thr Leu Lys Gly Arg Arg Leu                  85 90 95 Val Phe Asp Asn Gln Leu Thr Ile Lys Ser Pro Ser Lys Arg Glu Leu             100 105 110 Ala Lys Val His Gln Asn Lys Ile Leu Ser Ser Val Arg Lys Ser Gln         115 120 125 Glu Ile Thr Thr Asn Ser Glu Gln Arg Cys Pro Leu Lys Lys Glu Ser     130 135 140 Ala Cys Val Arg Leu Phe Lys Gln Glu Gly Thr Cys Tyr Gln Gln Ala 145 150 155 160 Lys Leu Val Leu Asn Thr Ala Val Pro Asp Arg Leu Pro Ala Arg Glu                 165 170 175 Arg Glu Met Asp Val Ile Arg Asn Phe Leu Arg Glu His Ile Cys Gly             180 185 190 Lys Lys Ala Gly Ser Leu Tyr Leu Ser Gly Ala Pro Gly Thr Gly Lys         195 200 205 Thr Ala Cys Leu Ser Arg Ile Leu Gln Asp Leu Lys Lys Glu Leu Lys     210 215 220 Gly Phe Lys Thr Ile Met Leu Asn Cys Met Ser Leu Arg Thr Ala Gln 225 230 235 240 Ala Val Phe Pro Ala Ile Ala Gln Glu Ile Cys Gln Glu Glu Val Ser                 245 250 255 Arg Pro Ala Gly Lys Asp Met Met Arg Lys Leu Glu Lys His Met Thr             260 265 270 Ala Glu Lys Gly Pro Met Ile Val Leu Val Leu Asp Glu Met Asp Gln         275 280 285 Leu Asp Ser Lys Gly Gln Asp Val Leu Tyr Thr Leu Phe Glu Trp Pro     290 295 300 Trp Leu Ser Asn Ser His Leu Val Leu Ile Gly Ile Ala Asn Thr Leu 305 310 315 320 Asp Leu Thr Asp Arg Ile Leu Pro Arg Leu Gln Ala Arg Glu Lys Cys                 325 330 335 Lys Pro Gln Leu Leu Asn Phe Pro Pro Tyr Thr Arg Asn Gln Ile Val             340 345 350 Thr Ile Leu Gln Asp Arg Leu Asn Gln Val Ser Arg Asp Gln Val Leu         355 360 365 Asp Asn Ala Ala Val Gln Phe Cys Ala Arg Lys Val Ser Ala Val Ser     370 375 380 Gly Asp Val Arg Lys Ala Leu Asp Val Cys Arg Arg Ala Ile Glu Ile 385 390 395 400 Val Glu Ser Asp Val Lys Ser Gln Thr Ile Leu Lys Pro Leu Ser Glu                 405 410 415 Cys Lys Ser Pro Ser Glu Pro Leu Ile Pro Lys Arg Val Gly Leu Ile             420 425 430 His Ile Ser Gln Val Ile Ser Glu Val Asp Gly Asn Arg Met Thr Leu         435 440 445 Ser Gln Glu Gly Ala Gln Asp Ser Phe Pro Leu Gln Gln Lys Ile Leu     450 455 460 Val Cys Ser Leu Met Leu Leu Ile Arg Gln Leu Lys Ile Lys Glu Val 465 470 475 480 Thr Leu Gly Lys Leu Tyr Glu Ala Tyr Ser Lys Val Cys Arg Lys Gln                 485 490 495 Gln Val Ala Ala Val Asp Gln Ser Glu Cys Leu Ser Leu Ser Gly Leu             500 505 510 Leu Glu Ala Arg Gly Ile Leu Gly Leu Lys Arg Asn Lys Glu Thr Arg         515 520 525 Leu Thr Lys Val Phe Phe Lys Ile Glu Glu Lys Glu Ile Glu His Ala     530 535 540 Leu Lys Asp Lys Ala Leu Ile Gly Asn Ile Leu Ala Thr Gly Leu Pro 545 550 555 560 <210> 2 <211> 442 <212> PRT <213> Homo sapiens <400> 2 Met Pro Gln Thr Arg Ser Gln Ala Gln Ala Thr Ile Ser Phe Pro Lys   1 5 10 15 Arg Lys Leu Ser Arg Ala Leu Asn Lys Ala Lys Asn Ser Ser Asp Ala              20 25 30 Lys Leu Glu Pro Thr Asn Val Gln Thr Val Thr Cys Ser Pro Arg Val          35 40 45 Lys Ala Leu Pro Leu Ser Pro Arg Lys Arg Leu Gly Asp Asp Asn Leu      50 55 60 Cys Asn Thr Pro His Leu Pro Pro Cys Ser Pro Pro Lys Gln Gly Lys  65 70 75 80 Lys Glu Asn Gly Pro Pro His Ser His Thr Leu Lys Gly Arg Arg Leu                  85 90 95 Val Phe Asp Asn Gln Leu Thr Ile Lys Ser Pro Ser Lys Arg Glu Leu             100 105 110 Ala Lys Val His Gln Asn Lys Ile Leu Ser Ser Val Arg Lys Ser Gln         115 120 125 Glu Ile Thr Thr Asn Ser Glu Gln Arg Cys Pro Leu Lys Lys Glu Ser     130 135 140 Ala Cys Val Arg Leu Phe Lys Gln Glu Gly Thr Cys Tyr Gln Gln Ala 145 150 155 160 Lys Leu Val Leu Asn Thr Ala Val Pro Asp Arg Leu Pro Ala Arg Glu                 165 170 175 Arg Glu Met Asp Val Ile Arg Asn Phe Leu Arg Glu His Ile Cys Gly             180 185 190 Lys Lys Ala Gly Ser Leu Tyr Leu Ser Gly Ala Pro Gly Thr Gly Lys         195 200 205 Thr Ala Cys Leu Ser Arg Ile Leu Gln Asp Leu Lys Lys Glu Leu Lys     210 215 220 Gly Phe Lys Thr Ile Met Leu Asn Cys Met Ser Leu Arg Thr Ala Gln 225 230 235 240 Ala Val Phe Pro Ala Ile Ala Gln Glu Ile Cys Gln Glu Glu Val Ser                 245 250 255 Arg Pro Ala Gly Lys Asp Met Met Arg Lys Leu Glu Lys His Met Thr             260 265 270 Ala Glu Lys Gly Pro Met Ile Val Leu Val Leu Asp Glu Met Asp Gln         275 280 285 Leu Asp Ser Lys Gly Gln Asp Val Leu Tyr Thr Leu Phe Glu Trp Pro     290 295 300 Trp Leu Ser Asn Ser His Leu Val Leu Ile Gly Ile Ala Asn Thr Leu 305 310 315 320 Asp Leu Thr Asp Arg Ile Leu Pro Arg Leu Gln Ala Arg Glu Lys Cys                 325 330 335 Lys Pro Gln Leu Leu Asn Phe Pro Pro Tyr Thr Arg Asn Gln Ile Val             340 345 350 Thr Ile Leu Gln Asp Arg Leu Asn Gln Val Ser Arg Asp Gln Val Leu         355 360 365 Asp Asn Ala Ala Val Gln Phe Cys Ala Arg Lys Val Ser Ala Val Ser     370 375 380 Gly Asp Val Arg Lys Ala Leu Asp Val Cys Arg Arg Ala Ile Glu Ile 385 390 395 400 Val Glu Ser Asp Val Lys Ser Gln Thr Ile Leu Lys Pro Leu Ser Glu                 405 410 415 Cys Lys Ser Pro Ser Glu Pro Leu Ile Pro Lys Arg Val Gly Leu Ile             420 425 430 His Ile Ser Gln Val Ile Ser Glu Val Asp         435 440 <210> 3 <211> 404 <212> PRT <213> Homo sapiens <400> 3 Met Pro Gln Thr Arg Ser Gln Ala Gln Ala Thr Ile Ser Phe Pro Lys   1 5 10 15 Arg Lys Leu Ser Arg Ala Leu Asn Lys Ala Lys Asn Ser Ser Asp Ala              20 25 30 Lys Leu Glu Pro Thr Asn Val Gln Thr Val Thr Cys Ser Pro Arg Val          35 40 45 Lys Ala Leu Pro Leu Ser Pro Arg Lys Arg Leu Gly Asp Asp Asn Leu      50 55 60 Cys Asn Thr Pro His Leu Pro Pro Cys Ser Pro Pro Lys Gln Gly Lys  65 70 75 80 Lys Glu Asn Gly Pro Pro His Ser His Thr Leu Lys Gly Arg Arg Leu                  85 90 95 Val Phe Asp Asn Gln Leu Thr Ile Lys Ser Pro Ser Lys Arg Glu Leu             100 105 110 Ala Lys Val His Gln Asn Lys Ile Leu Ser Ser Val Arg Lys Ser Gln         115 120 125 Glu Ile Thr Thr Asn Ser Glu Gln Arg Cys Pro Leu Lys Lys Glu Ser     130 135 140 Ala Cys Val Arg Leu Phe Lys Gln Glu Gly Thr Cys Tyr Gln Gln Ala 145 150 155 160 Lys Leu Val Leu Asn Thr Ala Val Pro Asp Arg Leu Pro Ala Arg Glu                 165 170 175 Arg Glu Met Asp Val Ile Arg Asn Phe Leu Arg Glu His Ile Cys Gly             180 185 190 Lys Lys Ala Gly Ser Leu Tyr Leu Ser Gly Ala Pro Gly Thr Gly Lys         195 200 205 Thr Ala Cys Leu Ser Arg Ile Leu Gln Asp Leu Lys Lys Glu Leu Lys     210 215 220 Gly Phe Lys Thr Ile Met Leu Asn Cys Met Ser Leu Arg Thr Ala Gln 225 230 235 240 Ala Val Phe Pro Ala Ile Ala Gln Glu Ile Cys Gln Glu Glu Val Ser                 245 250 255 Arg Pro Ala Gly Lys Asp Met Met Arg Lys Leu Glu Lys His Met Thr             260 265 270 Ala Glu Lys Gly Pro Met Ile Val Leu Val Leu Asp Glu Met Asp Gln         275 280 285 Leu Asp Ser Lys Gly Gln Asp Val Leu Tyr Thr Leu Phe Glu Trp Pro     290 295 300 Trp Leu Ser Asn Ser His Leu Val Leu Ile Gly Ile Ala Asn Thr Leu 305 310 315 320 Asp Leu Thr Asp Arg Ile Leu Pro Arg Leu Gln Ala Arg Glu Lys Cys                 325 330 335 Lys Pro Gln Leu Leu Asn Phe Pro Pro Tyr Thr Arg Asn Gln Ile Val             340 345 350 Thr Ile Leu Gln Asp Arg Leu Asn Gln Val Ser Arg Asp Gln Val Leu         355 360 365 Asp Asn Ala Ala Val Gln Phe Cys Ala Arg Lys Val Ser Ala Val Ser     370 375 380 Gly Asp Val Arg Lys Ala Leu Asp Val Cys Arg Arg Ala Ile Glu Ile 385 390 395 400 Val Glu Ser Asp <210> 4 <211> 324 <212> PRT <213> Homo sapiens <400> 4 Met Pro Gln Thr Arg Ser Gln Ala Gln Ala Thr Ile Ser Phe Pro Lys   1 5 10 15 Arg Lys Leu Ser Arg Ala Leu Asn Lys Ala Lys Asn Ser Ser Asp Ala              20 25 30 Lys Leu Glu Pro Thr Asn Val Gln Thr Val Thr Cys Ser Pro Arg Val          35 40 45 Lys Ala Leu Pro Leu Ser Pro Arg Lys Arg Leu Gly Asp Asp Asn Leu      50 55 60 Cys Asn Thr Pro His Leu Pro Pro Cys Ser Pro Pro Lys Gln Gly Lys  65 70 75 80 Lys Glu Asn Gly Pro Pro His Ser His Thr Leu Lys Gly Arg Arg Leu                  85 90 95 Val Phe Asp Asn Gln Leu Thr Ile Lys Ser Pro Ser Lys Arg Glu Leu             100 105 110 Ala Lys Val His Gln Asn Lys Ile Leu Ser Ser Val Arg Lys Ser Gln         115 120 125 Glu Ile Thr Thr Asn Ser Glu Gln Arg Cys Pro Leu Lys Lys Glu Ser     130 135 140 Ala Cys Val Arg Leu Phe Lys Gln Glu Gly Thr Cys Tyr Gln Gln Ala 145 150 155 160 Lys Leu Val Leu Asn Thr Ala Val Pro Asp Arg Leu Pro Ala Arg Glu                 165 170 175 Arg Glu Met Asp Val Ile Arg Asn Phe Leu Arg Glu His Ile Cys Gly             180 185 190 Lys Lys Ala Gly Ser Leu Tyr Leu Ser Gly Ala Pro Gly Thr Gly Lys         195 200 205 Thr Ala Cys Leu Ser Arg Ile Leu Gln Asp Leu Lys Lys Glu Leu Lys     210 215 220 Gly Phe Lys Thr Ile Met Leu Asn Cys Met Ser Leu Arg Thr Ala Gln 225 230 235 240 Ala Val Phe Pro Ala Ile Ala Gln Glu Ile Cys Gln Glu Glu Val Ser                 245 250 255 Arg Pro Ala Gly Lys Asp Met Met Arg Lys Leu Glu Lys His Met Thr             260 265 270 Ala Glu Lys Gly Pro Met Ile Val Leu Val Leu Asp Glu Met Asp Gln         275 280 285 Leu Asp Ser Lys Gly Gln Asp Val Leu Tyr Thr Leu Phe Glu Trp Pro     290 295 300 Trp Leu Ser Asn Ser His Leu Val Leu Ile Gly Ile Ala Asn Thr Leu 305 310 315 320 Asp Leu Thr Asp <210> 5 <211> 300 <212> PRT <213> Homo sapiens <400> 5 Met Pro Gln Thr Arg Ser Gln Ala Gln Ala Thr Ile Ser Phe Pro Lys   1 5 10 15 Arg Lys Leu Ser Arg Ala Leu Asn Lys Ala Lys Asn Ser Ser Asp Ala              20 25 30 Lys Leu Glu Pro Thr Asn Val Gln Thr Val Thr Cys Ser Pro Arg Val          35 40 45 Lys Ala Leu Pro Leu Ser Pro Arg Lys Arg Leu Gly Asp Asp Asn Leu      50 55 60 Cys Asn Thr Pro His Leu Pro Pro Cys Ser Pro Pro Lys Gln Gly Lys  65 70 75 80 Lys Glu Asn Gly Pro Pro His Ser His Thr Leu Lys Gly Arg Arg Leu                  85 90 95 Val Phe Asp Asn Gln Leu Thr Ile Lys Ser Pro Ser Lys Arg Glu Leu             100 105 110 Ala Lys Val His Gln Asn Lys Ile Leu Ser Ser Val Arg Lys Ser Gln         115 120 125 Glu Ile Thr Thr Asn Ser Glu Gln Arg Cys Pro Leu Lys Lys Glu Ser     130 135 140 Ala Cys Val Arg Leu Phe Lys Gln Glu Gly Thr Cys Tyr Gln Gln Ala 145 150 155 160 Lys Leu Val Leu Asn Thr Ala Val Pro Asp Arg Leu Pro Ala Arg Glu                 165 170 175 Arg Glu Met Asp Val Ile Arg Asn Phe Leu Arg Glu His Ile Cys Gly             180 185 190 Lys Lys Ala Gly Ser Leu Tyr Leu Ser Gly Ala Pro Gly Thr Gly Lys         195 200 205 Thr Ala Cys Leu Ser Arg Ile Leu Gln Asp Leu Lys Lys Glu Leu Lys     210 215 220 Gly Phe Lys Thr Ile Met Leu Asn Cys Met Ser Leu Arg Thr Ala Gln 225 230 235 240 Ala Val Phe Pro Ala Ile Ala Gln Glu Ile Cys Gln Glu Glu Val Ser                 245 250 255 Arg Pro Ala Gly Lys Asp Met Met Arg Lys Leu Glu Lys His Met Thr             260 265 270 Ala Glu Lys Gly Pro Met Ile Val Leu Val Leu Asp Glu Met Asp Gln         275 280 285 Leu Asp Ser Lys Gly Gln Asp Val Leu Tyr Thr Leu     290 295 300 <210> 6 <211> 204 <212> PRT <213> Homo sapiens <400> 6 Met Pro Gln Thr Arg Ser Gln Ala Gln Ala Thr Ile Ser Phe Pro Lys   1 5 10 15 Arg Lys Leu Ser Arg Ala Leu Asn Lys Ala Lys Asn Ser Ser Asp Ala              20 25 30 Lys Leu Glu Pro Thr Asn Val Gln Thr Val Thr Cys Ser Pro Arg Val          35 40 45 Lys Ala Leu Pro Leu Ser Pro Arg Lys Arg Leu Gly Asp Asp Asn Leu      50 55 60 Cys Asn Thr Pro His Leu Pro Pro Cys Ser Pro Pro Lys Gln Gly Lys  65 70 75 80 Lys Glu Asn Gly Pro Pro His Ser His Thr Leu Lys Gly Arg Arg Leu                  85 90 95 Val Phe Asp Asn Gln Leu Thr Ile Lys Ser Pro Ser Lys Arg Glu Leu             100 105 110 Ala Lys Val His Gln Asn Lys Ile Leu Ser Ser Val Arg Lys Ser Gln         115 120 125 Glu Ile Thr Thr Asn Ser Glu Gln Arg Cys Pro Leu Lys Lys Glu Ser     130 135 140 Ala Cys Val Arg Leu Phe Lys Gln Glu Gly Thr Cys Tyr Gln Gln Ala 145 150 155 160 Lys Leu Val Leu Asn Thr Ala Val Pro Asp Arg Leu Pro Ala Arg Glu                 165 170 175 Arg Glu Met Asp Val Ile Arg Asn Phe Leu Arg Glu His Ile Cys Gly             180 185 190 Lys Lys Ala Gly Ser Leu Tyr Leu Ser Gly Ala Pro         195 200 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 aaggatccct caaacccgat cccag 25 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> backward primer <400> 8 aaggatcctt aaggcaatcc ag 22 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for D404N <400> 9 gtagagtcaa atgtcaaaag c 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for D442N <400> 10 tcagaagtta atggtaacag g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for D502N <400> 11 gcggctgtga accagtcaga g 21 <210> 12 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for CDC6 <400> 12 aaggcctatg cctcaaaccc gatcccaggc acag 34 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CDC6 <400> 13 aactcgagtt aaggcaatcc agtagctaag at 32 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for CDC6 (1-442) <400> 14 aaggcctatg cctcaaaccc gatcccaggc acag 34 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CDC6 (1-442) <400> 15 aactcgagat caacttctga gatgacttgg g 31 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for CDC6 (111-560) <400> 16 aaggcctgaa ctagccaaag ttcac 25 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> Reverse primer for CDC6 (111-560) <400> 17 aactcgagtt aaggcaatcc agtagctaag at 32 <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for CDC6 primer <400> 18 aaggcctatg cctcaaaccc gatcccaggc acag 34 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CDC6 (1-100) <400> 19 aactcgagat tgtcaaatac caatcttcgt ccc 33 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CDC6 (1-204) <400> 20 aactcgagag gagcaccaga aaggtaaagg c 31 <210> 21 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CDC6 (1-300) <400> 21 aactcgagta gcgtgtacaa tacatcctgg cc 32 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CDC6 (1-324) <400> 22 aactcgagat ctgtgagatc cagggtatta gc 32 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CDC6 (1-404) <400> 23 aactcgagtt aatctgactc tacaatttca atagct 36

Claims (9)

An apoptosis inducing protein comprising amino acid sequences 1 to 179 of the CDC6 protein as set forth in SEQ ID NO: 1 and wherein amino acid sequences 462 to 488 are deleted. The protein of claim 1, wherein the protein further comprises a part or whole of amino acid sequence 180-442. 3. The protein of claim 2, wherein the protein consists of amino acid sequences 1 to 442 of the CDC6 protein set forth in SEQ ID NO: 2 . 3. The protein of claim 2, wherein the protein consists of amino acid sequences 1 to 404 of the CDC6 protein set forth in SEQ ID NO: 3 . 3. The protein of claim 2, wherein the protein consists of amino acid sequences 1 to 324 of the CDC6 protein set forth in SEQ ID NO: 4 . 3. The protein of claim 2, wherein the protein consists of amino acid sequences 1 to 300 of the CDC6 protein set forth in SEQ ID NO: 5 . 3. The protein of claim 2, wherein the protein consists of amino acid sequences 1 to 204 of the CDC6 protein set forth in SEQ ID NO: 6 . An apoptosis inducer containing the protein of any one of claims 1 to 7 as an active ingredient. 9. The apoptosis inducing agent according to claim 8, wherein the apoptosis inducing agent is used for preventing or treating various cancers or rheumatoid arthritis including breast cancer, rectal cancer and thyroid cancer.