KR20090103763A - Pharmaceutical composition containing CRIF1 protein or its fragment for cancer treatment - Google Patents

Abstract

The present invention relates to a CRIF1 protein or fragment thereof that functions to regulate DNA replication and genomic stabilization, DNA encoding the same, mRNA of the protein and a cancer prophylactic composition containing the same.

According to the present invention, by controlling the abnormal replication (cancer) of DNA using the protein CRIF1, in particular, the cc-domain that binds geminin, the DNA replication origin may be used for the prevention / inhibition and treatment of cancer.

Description

Pharmaceutical composition containing CRIF1 protein or its fragment for cancer treatment}

The present invention relates to a CRIF1 protein or fragment thereof that functions to regulate DNA replication and genomic stabilization and DNA encoding the same, and a cancer prevention-treatment composition containing mRNA of the protein.

Cell cycle regulation is a basic process for cell differentiation and growth. One cell cycle is a process in which DNA is replicated exactly once and cells are divided equally. The cell division process is performed according to the cell cycle progression of the G1-S-G2-M phase, which is controlled under strict control. If out of this control cell division abnormally will lead to instability of the genome and consequently cause cell death or the occurrence of cancer ^1,2.

In eukaryotic cells, DNA replication begins with the formation of a pre-replication complex (RC) at the end of somatic mitosis. pre-RC is formed, it is bonded to the origin of replication Orc (replication origin), and the combined sequence into DNA replication initiation factor of Cdc6 and Cdt1 and is made as a DNA replication helicase the MCM2-7 combined in a combination thereof ^1,3. The DNA is then replicated by CDK and various kinases, and the cell cycle divides into two daughter cells ⁴ . Cdt1, a replication license factor, is loaded and regulates DNA replication by inducing chromosome bonds such as MCM molecules. If Cdt1 is overexpressed, reloading is induced to induce rereplication. Becomes ^{5, 6} .

Geminin, the only Cdt1 inhibitor found in eukaryotic cells, interacts with Cdt1 to inhibit Cdt1 replication and consequently maintains genomic stabilization. geminin forms a dimer and then interacts with Cdt1 to form a geminin-Cdt1 complex. Therefore, the regulation of geminin dimer formation and the regulation of geminin-Cdt1 complex formation are very important for understanding the mechanism of DNA replication in eukaryotes ⁷ .

On the other hand, CR6 Interacting Factor 1 (CRIF 1) protein, which is found to increase in expression during cancer, is a protein that binds to CR6 (Gadd45gamma) protein. The mRNA of CRIF1 was not observed in peripheral blood mononuclear cells (PBMC) obtained from normal human body, whereas fetal kidney cell line, prostate cancer cell line, cervical cancer cell line, fibrosarcoma cell line, chorionic cancer cell line, It is known that its expression is increased in human blood cancer cell lines, HL-60, K-562, Jurkat, MOLT-4 and the like. Patent No. 439489 discloses a kit for diagnosing cancer using this characteristic of the CRIF1 gene. However, it is only known that the expression of CRIF1 is increased in cancer cells, and there is no known how CRIF1 itself functions in the DNA replication process and what effect it has.

Meanwhile, the coiled-coil domain (hereinafter, referred to as 'cc-domain') is known to play an important role in the interaction between proteins. The geminin, a DNA replication inhibitor, also has a cc-domain, which is critical for binding to Cdt1, the DNA replication initiator. It is known that CRIF1 binds to geminin, but it is also unknown whether cc-domains exist in CRIF1, whether cc-domains act on CRIF1 and gemini binding, and how their binding plays a role in DNA replication control. none.

If domain information directly involved in the binding of CRIF1 protein and geminin and the mechanism of DNA replication regulation are known, it can be effectively used for cancer prevention / inhibition and treatment by DNA replication regulation.

An object of the present invention is to utilize the CR-IF1, in particular ccin domains that bind to geminin to control the abnormal replication (cancer) of DNA to prevent cancer / suppression and treatment.

Another object of the present invention is to utilize the cc-domain function of geminin to control the physiological reactions and abnormal conditions controlled by geminin.

The present invention for achieving the above object relates to a pharmaceutical composition for the treatment of cancer comprising a CRIF 1 (CR6 Interacting Factor 1) protein or a fragment thereof represented by SEQ ID NO. will be. The fragment of the protein may be a cc-domain protein of CRIF1, ie, a cc-domain protein consisting of amino acids 172 to 212 of SEQ ID NO: 1 or a protein comprising a cc-domain.

The pharmaceutical composition for treating cancer may contain CRIF1 or a fragment thereof in the form of a protein, but may also contain a nucleic acid encoding the protein to express and produce CRIF1 in a cell. The composition also includes that the nucleic acid is contained in the expression vector.

The nucleic acid may be introduced into the target cell by various methods known in the art. For example, the nucleic acid may be introduced into an expression vector such as a plasmid or viral vector, and then the expression vector may be introduced into a cell by infection or transduction.

The pharmaceutical compositions of the present invention may be prepared by methods known in the pharmaceutical and food science fields, and may be mixed with themselves or with a pharmaceutically or food-acceptable carrier, forming agent, diluent, or the like. It can be prepared and used in various formulations.

The dosage of the pharmaceutical composition according to the present invention is naturally selected according to the age, sex and condition of the patient, the severity of the disease to be treated and the like.

The invention also relates to a cc-domain protein of CRIF1 represented by SEQ ID NO: 2, DNA, mRNA or PNA encoding said domain protein and a vector system containing the same. The cc-domain protein of CRIF1 is a domain protein that specifically interacts with geminin and is the domain protein responsible for the genomic stabilization function of CRIF1.

CRIF1 triggers cell cycle initiation, and this replication control function is induced by binding to geminin via the cc-domain at the C-terminus of the CRIF1 protein. According to the present invention as described above it is possible to control the DNA replication according to the cell cycle through the function control of geminin using the cc-domain of the CRIF1 protein and through this characteristic the treatment of diseases (eg cancer) showing cell cycle regulation abnormalities The effect can be obtained.

1 is a photograph showing that the CRIF1 protein interacts with geminin, a DNA replication factor.

2 is a diagram and graph showing the cc-domain prediction results of CRIF1 by SMART.

3 is a diagram and photograph identifying the CRIF1 domain interacting with geminin.

4 is a graph showing that overexpression of CRIF1 induces cell death of colon cancer cells.

5 is a graph showing that overexpression of CRIF1 inhibits cell proliferation of colorectal cancer cell lines.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and embodiments. However, these drawings and embodiments are only examples for easily explaining the contents and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. In addition, it will be apparent to those skilled in the art that various modifications and changes can be made within the scope of the present invention based on these examples.

The terms, techniques, and the like described in this specification are used in the meanings generally used in the art to which the present invention pertains, unless otherwise specified. In addition, all the documents mentioned in this specification are included in this specification as the documents for demonstrating this invention.

Example

Example 1 Identification of Factors and Sites Interacting with CRIF1

(One) Identification of Factors Interacting with CRIF1

The interaction of CRIF1 with DNA replication factors was confirmed by yeast two hybrid assay ⁸ .

In more detail, the cDNA libraries of pLexA-CRIF1 (Clontech Labotatories) and K-562 cell lines (Clontech Laboratories, Inc) were introduced into Y190 yeast (Clontech Labotatories, Inc). They were then incubated overnight in SD selective cultures without histidine, leucine, tryptophan, and 3-amino-1,2,4-triazole (Sigma) added at 45 mM, and 72 blue colonies were selected by galactosidase filter assay.

Galactosidase filter assay was carried out by the following method. In other words, Z buffer (60 mM Na2HPO4, Sterilized 1 mM MgSO4, 60 mM NaH2PO4, 10 mM KCl, Sigma, respectively), 2% X-Gal (DUCHEFA) and 2-Mercaptoethanol (Sigma) were mixed and then dispensed in Petri dishes. Cover the Watman filter (Watman). Take yeast replicas incubated in a plate with Whatman filter and soak them in liquid nitrogen for 10 seconds. The watman filter removed from the liquid nitrogen was carefully transferred to a Petri dish containing the first mixed solution, incubated overnight at 37 ° C. incubator, and the blue colony was selected.

To identify the interacting factors, yeasts of blue colony were grown on SD medium without histidine, leucine, and tryptophan, and then DNA was extracted. Thereafter, HB101 Escherichia coli (Takara) was transformed to obtain a large amount of DNA, and each factor was identified through sequencing. As a result, the protein binding to CRIF1 was found to be geminin, an important factor for DNA replication.

To verify CRIF1 and geminin binding intracellularly, Adv-CRIF1 adenovirus was overexpressed in HSF cells and immunoprecipitated with anti-geminin antibody (Santa Cruze Inc.) and Western blot performed to bind CRIF1 and geminin. Reconfirmed.

First, a vector expressing the CRIF1-GFP protein was produced using the following method. From the CRIF1 full-length DNA of SEQ ID NO: 3 using the primers of SEQ ID NOs: 4 and 5, the nucleotide sequences recognizable by the restriction enzymes XhoI and EcoRI are linked to the N-terminus and C-terminus of the DNA sequence encoding the CRIF1 protein. Amplified by PCR and cleaved with XhoI and EcoRI (TOYOBO) to obtain a CRIF1 insertion sequence. The CRIF1 full-length DNA was extracted from the HeLa cell line by using RT-PCR method using EX-Taq (TkKaRa) and transferred to pGEM T-Easy vector (Promega).

SEQ ID NO: 4: ACTCGAGATGGCGGCGTCCGTG (Forward)

SEQ ID NO: 5 CCCCGTGGGTCGAGGGCTTAAGT (Reverse)

The CRIF1 insertion sequence obtained by the above method was ligation into the pShuttle-CMV vector (Qbiogene, Inc). The prepared shuttle vector was cotransfected into a 293T cell line (ATCC) with a pJM17 viral vector (Qbiogene, Inc), followed by incubation in a CO 2 incubator at 37 ° C, and observed plaque production. When plaque production was confirmed, the culture medium containing the vrirus was collected, infected with 293T cell line in a large culture, and the virus was extracted.

 In order to confirm the binding of CRIF1 and geminin, Adv-CRIF1 prepared in HSF cell line was infected and incubated for 48 hours in a 37 ° C. CO2 incubator. After washing the cells twice with PBS, cells were lysed using RIPA buffer. Cell lysates were centrifuged at 13000 rpm for 15 minutes using a centrifuge, and then the supernatant was taken to measure protein concentration by bradford (BioRad) method. GFP antibody (Santacruz) was added to 500 μg of protein at a ratio of 1/100 and stirred at 4 ° C. for 4 hours. After 4 hours, 30 μl of Protein A / G PLUS-Agarose Immunoprecipitation reagent (Santacruze) was added and immunoprecipitated overnight at 4 ° C. After stirring, the supernatant was removed and washed with fresh RIPA buffer. This washing process was carried out five times to remove the last supernatant, and then placed in 2X sds sample buffer and heated for 5 minutes at 100 ℃ boiling water to prepare a sample, electrophoresis on a polyacrylamide gel. The electrophoresis gel was blocked on nitrocellulose membrane (Amersham Bioscience) and then blocked for 1 hour in 5% skim milk. Since the first antibody to detect geminin was added and reacted with O / N at 4 ℃. After reacting with the primary antibody, it was washed three times with TBS / T for 10 minutes and again with rabbit secondary antibody (CELL SIGNALING) for 1-3 hours. After reacting with the secondary antibody, it was washed three times with TBS / T for 10 minutes and detected on an x-ray photosensitive film (FUJIFILM) using ECL solution (iNtRON). As shown in FIG. 1 showing the results of the western blot experiment, it was found that CRIF1 and geminin specifically interacted with each other.

(2) CRIF1 domain mapping to interact with geminin

Coiled coil (cc) domains are known to play a role in the interaction between proteins, and geminin is also known to have coiled coil domains. There is also a cc-domain in CRIF1, so interaction with giminin is likely through this cc-domain. To verify this, the existence of cc-domain in CRIF1 was tested using the cc-domain prediction program (SMART). As a result, it was found that CRIF1 also has a coiled coil domain and its position is amino acid sequence 172-212 (FIG. 2).

In order to confirm the interaction between geminin and CRIF1 and to find the specific domain of CRIF1 that interacts with geminin, the coiled coil (cc) domain of CRIF1 was cut using pCMV-Tag2 (Strata gene Inc.) vector. CRIF1ΔCC, CRIF1 △ N c-deletion mutant and wild type CRIF1 to express the protein of the truncated form (99-171 of SEQ ID NO: 1) or the N-terminus Produced.

Flag-CRIF1 wt plasmid DNA which expresses wild type CRIF1 was amplified by PCR method from CRIF1 full-length DNA of SEQ ID NO: 3 using primers of SEQ ID NOs: 6 and 7, and cleaved by restriction enzymes BamHI, XhoI (TOYOBO) It was then ligated to the pCMV-Tag2 vector (Stratagene).

SEQ ID NO: 6 GGATCCATGGCGGCGTCCGTG (Forward)

SEQ ID NO: 7 CTCGAGGGGCACCCAGCTCCTGA (Reverse)

Construction of Flag-CRIF1ΔCC plasmid DNA, which allows the cc domain of CRIF1 to express the truncated form of CRIF1, was amplified by PCR from CRIF1 full-length DNA of SEQ ID NO: 3 using primers SEQ ID NOs: 6 and 8 BamHI, XhoI (TOYOBO) was cut and ligated to the pCMV-Tag2 vector (Stratagene).

SEQ ID NO: 6 GGATCCATGGCGGCGTCCGTG (Forward)

SEQ ID NO: CTCGAGTCACCTTGGGTCCAC (Reverse)

Flag-CRIF1ΔN plasmid DNA, which causes the N-terminus of CRIF1 to express the truncated form of CRIF1, was amplified by PCR from CRIF1 full-length DNA of SEQ ID NO: 3 using primers of SEQ ID NOs: 9 and 7, followed by restriction enzyme BamHI. , XhoI (TOYOBO) was cut and then ligated to the pCMV-Tag2 vector (Stratagene) was produced.

SEQ ID NO: GGATCCATGCAGGAGTCGCTG (Forward)

SEQ ID NO: 7 CTCGAGGGGCACCCAGCTCCTGA (Reverse)

Flag-CRIF1 wt, Flag-CRIF1ΔCC, and Flag-CRIF1ΔN plasmid DNA were transfected into Lipofectamin PLUS (invitrogen) in Hela cell lines, and then cultured in a CO 2 incubator for 24 hours at 37 ° C. After washing the cells twice with PBS, cells were lysed using RIPA buffer. Cell lysates were centrifuged at 13000 rpm for 15 minutes using a centrifuge, and then the supernatant was taken to measure protein concentration by bradford (BioRad) method. Flag antibody (stratagene) was added to 500 ug of protein at a ratio of 1/100 and stirred at 4 ° C. for 4 hours. After 4 hours, 30 μl of Protein A / G PLUS-Agarose Immunoprecipitation reagent (Santacruze) was added and immunoprecipitated overnight at 4 ° C. After stirring, the supernatant was removed and washed with fresh RIPA buffer. This washing process was carried out five times, and after removing the last supernatant, 2X sds sample buffer was added and heated in 100 ° C. boiling water for 5 minutes. The samples made were electrophoresed on polyacrylamide gels. The electrophoresis gel was blocked on nitrocellulose membrane (Amersham Bioscience) and then blocked for 1 hour in 5% skim milk. Since the first antibody to detect geminin was added and reacted with O / N at 4 ℃. After reacting with the primary antibody, it was washed three times with TBS / T for 10 minutes and again with rabbit secondary antibody (CELL SIGNALING) for 1-3 hours. After reacting with the secondary antibody, it was washed three times with TBS / T for 10 minutes and detected on an x-ray photosensitive film (FUJIFILM) using ECL solution (iNtRON) (FIG. 3).

As a result, as shown in FIG. 3, when CRIF1 wt and CRIF1ΔN were transformed, specific bands with geminin could be identified, and when CRIF1 △ CC was transformed, interaction with geminin could not be confirmed. . This shows that the cc-domain of CRIF1 is an important domain for interaction with geminin.

Example 2 Confirmation of Inhibition of Cancer Cell Proliferation by CRIF1

As it was confirmed that CRIF1 interacts with geminin, which acts as a genome stabilizing function, the effects of CRIF1 on the cell cycle and cell proliferation of cancer cells were identified.

Adv-CRIF1, a CRIF 1 adenovirus prepared in Example 1, was infected with cultured colorectal cancer cell line HCT116 (ATCC), and then cultured in a CO 2 incubator at 37 ° C. for 24 hours. Then, DNA content was observed through FACS (BD FACSCanto ™ flow cytometer, BD bioscience) analysis, and the results are shown in FIG. 4.

From FIG. 4, when CRIF1 is overexpressed in HCT116 (ATCC), the colorectal cancer cell line, the number of cells in the G1 phase is decreased and the number of cells in the sub G1 section indicating apoptosis is increased.

In addition, after overexpressing CRIF1 in the HCT116 cell line, the number of cells was measured by Trypan blue exclusion assay method at 24 hour intervals after 24 hours. As a result, the proliferation of cells was significantly slower than that of the control group (FIG. 5). From this, it was confirmed that CRIF1 can inhibit cancer cell proliferation by regulating the cell cycle of cancer cells.

references

Bell, S. P. & Dutta, A. DNA replication in eukaryotic cells. Annu Rev Biochem 71, 333-74 (2002).

Hartwell, L. H. & Kastan, M. B. Cell cycle control and cancer. Science 266, 1821-8 (1994).

Bell, S. P. & Stillman, B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357, 128-34 (1992).

4.Waga, S. & Stillman, B. The DNA replication fork in eukaryotic cells. Annu Rev Biochem 67, 721-51 (1998).

5.Mororano, D., Moreau, J. & Mechali, M. XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Nature 404, 622-5 (2000).

6.Nishitani, H., Lygerou, Z., Nishimoto, T. & Nurse, P. The Cdt1 protein is required to license DNA for replication in fission yeast. Nature 404, 625-8 (2000).

7. Wohlschlegel, J. A. et al. Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Science 290, 2309-12 (2000).

8. Chung, H. K. et al. CR6-interacting factor 1 interacts with Gadd45 family proteins and modulates the cell cycle. J Biol Chem 278, 28079-88 (2003).

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Pharmaceutical composition containing CRIF1 protein or its fragment for <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 222 <212> PRT <213> CIRF1 protein <400> 1 Met Ala Ala Ser Val Arg Gln Ala Arg Ser Leu Leu Gly Val Ala Ala 1 5 10 15 Thr Leu Ala Pro Gly Ser Arg Gly Tyr Arg Ala Arg Pro Pro Pro Arg 20 25 30 Arg Arg Pro Gly Pro Arg Trp Pro Asp Pro Glu Asp Leu Leu Thr Pro 35 40 45 Arg Trp Gln Leu Gly Pro Arg Tyr Ala Ala Lys Gln Phe Ala Arg Tyr 50 55 60 Gly Ala Ala Ser Gly Val Val Pro Gly Ser Leu Trp Pro Ser Pro Glu 65 70 75 80 Gln Leu Arg Glu Leu Glu Ala Glu Glu Arg Glu Trp Tyr Pro Ser Leu 85 90 95 Ala Thr Met Gln Glu Ser Leu Arg Val Lys Gln Leu Ala Glu Glu Gln 100 105 110 Lys Arg Arg Glu Arg Glu Gln His Ile Ala Glu Cys Met Ala Lys Met 115 120 125 Pro Gln Met Ile Val Asn Trp Gln Gln Gln Gln Arg Glu Asn Trp Glu 130 135 140 Lys Ala Gln Ala Asp Lys Glu Arg Arg Ala Arg Leu Gln Ala Glu Ala 145 150 155 160 Gln Glu Leu Leu Gly Tyr Gln Val Asp Pro Arg Ser Ala Arg Phe Gln 165 170 175 Glu Leu Leu Gln Asp Leu Glu Lys Lys Glu Arg Lys Arg Leu Lys Glu 180 185 190 Glu Lys Gln Lys Arg Lys Lys Glu Ala Arg Ala Ala Ala Leu Ala Ala 195 200 205 Ala Val Ala Gln Asp Pro Ala Ala Ser Gly Ala Pro Ser Ser 210 215 220 <210> 2 <211> 40 <212> PRT <213> cc-domain protein of CRIF1 <400> 2 Ala Arg Phe Gln Glu Leu Leu Gln Asp Leu Glu Lys Lys Glu Arg Lys 1 5 10 15 Arg Leu Lys Glu Glu Lys Gln Lys Arg Lys Lys Glu Ala Arg Ala Ala 20 25 30 Ala Leu Ala Ala Ala Val Ala Gln 35 40 <210> 3 <211> 666 <212> DNA <213> CRIF1 <400> 3 atggcggcgt ccgtgcgaca ggcacgcagc ctactaggtg tggcggcgac cctggccccg 60 ggttcccgtg gctaccgggc gcggccgccc ccgcgccgca ggccgggacc ccggtggcca 120 gaccccgagg acctcctgac cccgcggtgg cagctgggac cgcgctacgc ggctaagcag 180 ttcgcgcgtt acggcgccgc ctccggggtg gtccccggtt cgttatggcc gtcgccggag 240 cagctgcggg agctggaggc cgaagaacgc gaatggtacc cgagcctggc gaccatgcag 300 gagtcgctgc gggtgaagca gctggccgaa gagcagaagc gtcgggagag ggagcagcac 360 atcgcagagt gcatggccaa gatgccacag atgattgtga actggcagca gcagcagcgg 420 gagaactggg agaaggccca ggctgacaag gagaggaggg cccgactgca ggctgaggcc 480 caggagctcc tgggctacca ggtggaccca aggagtgccc gcttccagga gctgctccag 540 gacctagaga agaaggagcg caagcgcctc aaggaggaaa aacagaaacg gaagaaggag 600 gcgcgagctg ctgcattggc tgcagctgtg gctcaagacc cagcagcctc tggggcaccc 660 agctcc 666 <210> 4 <211> 22 <212> DNA <213> forward primer <400> 4 actcgagatg gcggcgtccg tg 22 <210> 5 <211> 23 <212> DNA <213> reverse primer <400> 5 ccccgtgggt cgagggctta agt 23 <210> 6 <211> 21 <212> DNA <213> forward primer <400> 6 ggatccatgg cggcgtccgt g 21 <210> 7 <211> 23 <212> DNA <213> reverse primer <400> 7 ctcgaggggc acccagctcc tga 23 <210> 8 <211> 21 <212> DNA <213> forward primer <400> 8 ctcgagtcac cttgggtcca c 21 <210> 9 <211> 21 <212> DNA <213> reverse primer <400> 9 ggatccatgc aggagtcgct g 21

Claims (10)

A pharmaceutical composition for treating cancer, comprising a CRIF 1 (CR6 Interacting Factor 1) protein or a fragment thereof represented by SEQ ID NO: 1 as an active ingredient. The method of claim 1, The fragment is a pharmaceutical composition for treating cancer comprising amino acids 172 to 212 of SEQ ID NO: 1. The method of claim 1, The fragment is a cc-domain protein represented by SEQ ID NO: 2 pharmaceutical composition for cancer treatment. A pharmaceutical composition for treating cancer comprising a nucleic acid encoding a CRIF 1 protein or a fragment thereof represented by SEQ ID NO: 1 as an active ingredient. The method of claim 4, wherein The fragment is a pharmaceutical composition for treating cancer comprising amino acids 172 to 212 of SEQ ID NO: 1. The method of claim 4, wherein A pharmaceutical composition for treating cancer, wherein the fragment is a cc-domain protein of CRIF 1 represented by SEQ ID NO: 2. The method according to any one of claims 4 to 6, Pharmaceutical composition, characterized in that the nucleic acid is contained in the expression vector. The cc-domain protein of CRIF1 represented by SEQ ID NO: 2, which functions for DNA replication regulation and genomic stabilization. DNA, mRNA or PNA encoding the domain protein of claim 8. A vector system containing the DNA according to claim 9.