KR100434591B1 - Human cancer suppressor gene, protein encoded therein, expression vector containing same, and cell transformed by said vector - Google Patents

Abstract

The present invention relates to a human cancer suppressor gene having the nucleotide sequence of SEQ ID NO: 1, a protein encoded therewith, an expression vector comprising the same, and a microorganism transformed with the vector. It can be usefully used for treatment.

Description

HUMAN CANCER SUPPRESSOR GENE, PROTEIN ENCODED THEREIN, EXPRESSION VECTOR CONTAINING SAME, AND CELL TRANSFORMED BY SAID VECTOR}

The present invention relates to human cancer suppressor genes, proteins encoded thereby, expression vectors comprising the same, and cells transformed with the vectors.

Cancer suppressor gene products inhibit transformation from normal cells to cancer cells, and loss of this function leads to malignant transformation (Klein, G., FASEB J. , 7 , 821- 825 (1993). In order for cancer cells to form cancer, normal copy number function of cancer suppressor genes must be lost. Modifications in the coding sequence of the p53 cancer suppressor gene have been found to be the most common genetic changes in human cancers (Bishop, JM, Cell , 64 , 235-248 (1991); and Weinberg, RA, Science , 254 , 1138-1146 ( 1991).

However, the reported p53 variation in cervical cancer ranges from only 2% to 11% (Crook, T. et al., Lancet , 339 , 1070-1073 (1992); and Busby-Earle, RMC et al., Br j. Cancer , 69 , 732-737 (1994)), and only a fraction of cervical cancer tissues are thought to exhibit this p53 mutation.

Accordingly, the present inventors have used a new mRNA differential display (DD) method to more efficiently express genes that are differentially expressed between normal cervical tissue and cervical cancer. The study was carried out to isolate.

An object of the present invention is to provide a novel human cancer suppressor gene.

Another object of the present invention is to provide a cancer suppressor protein encoded by the gene.

It is another object of the present invention to provide an expression vector comprising the gene.

Another object of the present invention to provide a cell transformed with the vector.

1 is a gel photograph showing PCR results using 5'13mer optional primer H-AP37 of SEQ ID NO: 3 and a fixed oligo-dT primer of SEQ ID NO: 4. FIG.

Figure 2 is a photograph showing the SDS-PAGE analysis of the gene product of the present invention.

FIG. 3A is a northern blot result showing differential expression levels of HCCS-4 gene in normal cervical tissue, primary cervical cancer tissue and cervical cancer cell line, and FIG. 3B is a northern blot result of hybridizing the same blot with β-actin probe .

Figure 4a is a northern blot result showing the differential expression level of the HCCS-4 gene in several normal tissues, Figure 4b is a northern blot result of hybridizing the same blot with β-actin probe.

Figure 5a is a northern blot result showing the differential expression level of HCCS-4 gene in several cancer cell lines, Figure 5b is a northern blot result of hybridizing the same blot with β-actin probe.

Figure 6 is a growth curve of wild type HeLa cells, HeLa cervical cancer cells transfected with the gene of the present invention and HeLa cells transfected with the expression vector N-terminal FLAG.

In accordance with the above object, the present invention provides a human cancer suppressor gene (named HCCS-4) having a nucleotide sequence of SEQ ID NO: 1.

According to this another object, the present invention provides a human cancer inhibiting protein having an amino acid sequence of SEQ ID NO: 2.

According to another object, the present invention provides an expression vector comprising the gene.

According to another object, the present invention provides a cell transformed with the vector.

Hereinafter, the present invention will be described in detail.

The gene of the present invention is human cancer suppressor gene 4 (HCCS-4) having a nucleotide sequence of SEQ ID NO: 1, wherein the base sequence of SEQ ID NO: 1 is registered in the NIH GenBank database of the National Institutes of Health No. AF465843 It is a new gene that is registered as an issue (published: February 1, 2003) and has little similarity with other sequences registered in this database.

The base sequence of SEQ ID NO: 1 includes one open reading frame corresponding to base numbers 170 to 1108 (base numbers 1106-1108 are end codons). However, due to the degeneracy of the codons or taking into account the codons preferred in the organism in which the genes are to be expressed, the genes of the present invention do not change the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region. Various modifications can be made to the various modifications or modifications can be made within a range that does not affect the expression of the gene in parts other than the coding region, such modified genes are also included in the scope of the invention. Accordingly, the present invention also encompasses polynucleotides having fragment sequences that are substantially identical to those of the genes. By substantially identical polynucleotides are meant those having sequence homology of at least 80%, preferably at least 90% and most preferably at least 95%.

The protein expressed from the gene of the present invention consists of 312 amino acid residues, has an amino acid sequence of SEQ ID NO: 2, and is about 35 kDa in size. However, even in the amino acid sequence of the protein, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Thus, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as said protein, wherein substantially identical polypeptides have at least 80%, preferably at least 90%, most preferably at least 95% sequence homology. It means things that have.

The genes and proteins of the present invention may be isolated from human tissues or synthesized according to known DNA or peptide synthesis methods. For example, the gene of the present invention can be obtained by screening and cloning according to a conventional method based on the nucleotide sequence information of SEQ ID NO: 1. As another example, reverse transcription using normal primers, and any primer H-AP37 of SEQ ID NO: 3 and an anchored oligo-dT primer of SEQ ID NO: 4 for total RNA extracted from cancer tissue or cell lines Perform reverse transcription-polymerase chain reaction (RT-PCR) to obtain 278 bp cDNA fragments that are differentially expressed in normal tissues but not in cancer tissues or cell lines, and used as probes to cDNA Full length cDNA clones can be obtained by plaque hybridization with the library.

The gene thus prepared is inserted into a microorganism or animal cell expression vector known in the art to prepare an expression vector, and then introduced into an appropriate host cell, for example, E. coli or HeLa cell line, thereby replicating a large amount of DNA of the gene. Or mass-produce proteins. In the production of the vector, expression control sequences such as promoters, terminators, self-replicating sequences, secretion signals, etc. may be appropriately selected and combined according to the type of host cell to produce the gene or protein.

We inserted HCCS-4 full-length cDNA into the expression vector pCEV-LAC (Miki, T. et al., Gene , 83 , 137-146 (1989)) and transformed E. coli DH5α, and obtained transformants Was named Escherichia coli DH5α / HCCS-4 / pCEV-LAC and was deposited with KCTC 10178BP dated February 18, 2002 to Gene Bank of Korea Biotechnology Research Institute.

The genes of the present invention appear to be overexpressed in normal tissues, preferably liver, kidney, placenta, thymus, spleen, skeletal muscle and heart tissues to inhibit carcinogenesis. In these tissues, the genes of the present invention are mostly overexpressed with about 1 kb of mRNA transcript. In particular, the gene of the present invention is differentially expressed only in normal tissues, for example, in cancer tissues and cells such as cervical cancer tissue, metastatic common iliac lymph node tissue, and cervical cancer cell line CUMC-6. It is differentially expressed only in normal cervical tissue.

Since the cancer cell line into which the gene of the present invention is introduced has a high killing rate, the gene of the present invention can be usefully used for the prevention and treatment of cancer.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Reference Example: Isolation of Total RNA

Total RNA was isolated from fresh tissue or cultured cells using a RNeasy total RNA kit (Qiagen Inc., Germany), followed by a message clean kit (GenHunter Corp., MA, USA). To remove DNA contaminated with RNA.

Example 1 Isolation and mRNA Differentiation of Total RNA

Differentiation patterns of gene expression in normal cervical tissue, primary cervical cancer tissue and cervical cancer cell lines were investigated as follows.

Normal exocervical tissue samples were obtained from hysterectomy patients during hysterectomy, and primary cervical cancer tissues and metastatic cartilage during radical hysterectomy in patients who did not receive radiation or chemotherapy prior to surgical treatment. Lymph node tissue samples were obtained. Human cervical cancer cell lines CUMC -6 (Kim, JW et al., Gynecol. Oncol. , 62 , 230-240 (1996)) and HeLa cervical cancer cell lines were used. From these tissues and cells, total RNA was isolated in the same manner as in the reference example.

Using total RNA samples from each tissue and cell, Liang, P. and Pardee, AB, Science , 257 , 967-971 (1992); and Liang, P. et al., Cancer Res. , 52 , 6966- 6968 (1993)) was modified in part to perform RT-PCR as follows. 0.2 μg of total RNA was reverse transcribed using the kit (RNAimage kit, GenHunter) with the H-T11A fixed oligo-dT primer of SEQ ID NO: 4, followed by the same fixed oligo-dT primer and 5 ′ of SEQ ID NO: 3 PCR was performed in the presence of 0.5 mM [α- ³⁵ S] -labeled dATP (1,200 Ci / mmol) using the 13mer optional primer H-AP37 (RNAimage primer set 1, GenHunter Corporation, USA). PCR was repeated 40 times at 40 ° C. at 40 ° C., 2 minutes at 40 ° C., and 40 seconds at 72 ° C., followed by reaction at 72 ° C. for 5 minutes. The amplified fragments were electrophoresed on electrophoresis on 6% polyacrylamide sequencing gels.

FIG. 1 shows PCR results using 5'13mer optional primer H-AP37 of SEQ ID NO: 3 and a fixed oligo-dT primer of SEQ ID NO: 4. FIG. In Figure 1, the first row is normal cervical tissue, the second row is cervical cancer tissue, the third row is metastatic iliac lymph node tissue, and the fourth row is cervical cancer cell line CUMC-6. As shown in FIG. 1, 278 bp cDNA fragments which were differentially expressed only in normal cervical tissue but not expressed in cervical cancer tissue, metastatic iliac lymph node tissue and cervical cancer cell line CUMC-6 were identified. This cDNA fragment was named CG375.

Cut the 278 bp band, the CG375 fragment, from the dried gel and boil for 15 minutes to elute the cDNA, then use the same primer pairs as above without using [α- ³⁵ S] -labeled dATP and 20 μM dNTP. PCR was performed under the same conditions and reamplified. The re-amplified cDNA fragment CG375 was cloned into the expression vector pGEM-T Easy using a cloning system (TA cloning system, Promega), and the base was determined using a sequencing kit (Sequenase Version 2.0 DNA Sequencing System, United States Biochemical Co.). The sequence was determined. cDNA fragment CG375 shows the nucleotide sequence of SEQ ID NO: 5.

The base sequence of SEQ ID NO: 5 was searched in the NIH GenBank database of the National Institutes of Health using the BLAST and FASTA programs, and showed little similarity with the base sequence registered in this database.

Example 2: cDNA Library Screening

The cDNA fragment CG375 obtained in Example 1 was labeled according to the method of Feinberg, AP and Vogelstein, B., Anal. Biochem. , 132 , 6-13 (1983) to obtain a ³² P-labeled CG375 cDNA probe. Bacteriophage lambdagt11 human lung embryonic fibroblast cDNA library (Miki, T. et al., Gene , 83 , 137-146 (1989)) and Sambrook, J. et al., Molecular Cloning : Plaque hybridization was carried out according to the method of A Laboratory mannual, New York: Cold Spring Harbor Laboratory (1989) to obtain a full-length cDNA clone of the human cervical cancer suppressor gene HCCS-4.

The nucleotide sequence of this full-length cDNA was determined, and the result was as SEQ ID NO: 1. This base sequence includes an open reading frame encoding 312 amino acid residues, the amino acid sequence deduced therefrom from SEQ ID NO: 2. The molecular weight of the inferred protein was also about 35 kDa.

The obtained HCCS-4 full-length cDNA clone was inserted into the prokaryotic expression vector pCEV-LAC (Miki, T. et al., Gene , 83 , 137-146 (1989)) and transformed into E. coli DH5α. The transformant was named Escherichia coli DH5α / HCCS-4 / pCEV-LAC and was deposited with KCTC 10178BP dated February 18, 2002 to the Gene Bank of Korea Research Institute of Bioscience and Biotechnology.

In addition, the expression vector pCEV-LAC into which the HCCS-4 full-length cDNA was inserted was digested with Sal I to obtain HCCS-4 full-length cDNA, and then the Sal I site of the prokaryotic expression vector pGEX 4T-3 (Amersham Pharmacia Biotech., USA). E. coli BL21 was transformed with the obtained vector after insertion. The transformed Escherichia coli was cultured in LB broth, and then 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture and reacted at 37 ° C. for 3 hours to express the HCCS-4 gene. . SDS-PAGE was performed by obtaining a protein sample from this culture according to the method of Sambrook, J. et al., Molecular Cloning: A Laboratory mannual, New York: Cold Spring Harbor Laboratory (1989).

Figure 2 is a photograph showing the results of SDS-PAGE analysis of HCCS-4 protein. In FIG. 2, the first column is a protein sample before induction by IPTG, and the second column is a protein sample after inducing expression of HCCS-4 gene by IPTG. As shown in Figure 2, the size of the expressed HCCS-4 protein is about 35 kDa, which is consistent with the inference from the base sequence.

Example 3: Northern Blot of HCCS-4 Gene

In order to investigate the expression level of HCCS-4 gene, Northern blot was performed as follows.

Three normal cervical tissue samples obtained in Example 1, three primary cervical cancer tissue samples, and 20 µg of total RNA of the cervical cancer cell lines HeLa (ATCC CCL-2) and CUMC-6 were denatured, respectively, and then 1% formaldehyde agar. Electrophoresis on Ross gels followed by transfer to nylon membrane (Boehringer-Mannheim, Germany). The nylon membrane was then hybridized overnight at 42 ° C. with ³² P-labeled random prime probes prepared from HCCS-4 full length cDNA using the Rediprime II random prime labeling system (Amersham, UK). The Northern blot process was repeated twice in the same manner, one was quantified by a densitometer, and the other was hybridized with β-actin probe to confirm the total amount of mRNA.

FIG. 3A is a Northern blot result showing the differential expression levels of HCCS-4 gene in normal cervical tissue, primary cervical cancer tissue and cervical cancer cell lines HeLa (Clontech) and CUMC-6, and FIG. 3B shows the same blot β-actin probe Northern blot results. As shown in Figures 3a and 3b, the expression level of HCCS-4 gene was high in all three cervical tissue samples, and the expression level of the three cervical cancer tissue samples was lower than that of normal tissues. It did not appear in two samples.

Northern blots were performed on normal human multiple tissue (Clontech) and human cancer cell lines (Clontech). In other words, normal tissues include normal brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocyte tissues, and cancer cell lines include promyelocytic leukemia HL-60, HeLa cervical cancer cells, Blots transfected with total RNA samples extracted from chronic myeloid leukemia cell line K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, SW480 colon cancer cells, A549 lung cancer cells, and G361 melanoma cells were cloned. Clontech, USA) was hybridized in the same manner to perform Northern blot.

Figure 4a is a northern blot result showing the differential expression level of the HCCS-4 gene in several normal tissues, Figure 4b is a northern blot result of hybridizing the same blot with β-actin probe. As shown in FIG. 4A, approximately 1 kb of dominant HCCS-4 mRNA transcript was overexpressed in liver, kidney, placenta, thymus, spleen, skeletal muscle and heart tissues.

Figure 5a is a northern blot result showing the differential expression level of HCCS-4 gene in several cancer cell lines, Figure 5b is a northern blot result of hybridizing the same blot with β-actin probe. As shown in Figure 5a, weakly expressed in Burkitt's lymphoma large and lymphoblastic leukemia MOLT-4, other myeloid leukemia HL-60, HeLa cervical cancer cells, chronic myeloid leukemia cell lines K-562, SW480 colon cancer cells, A549 lung cancer cells , But no HCCS-4 gene was expressed in G361 melanoma cells. These results indicate that the HCCS-4 gene of the present invention has tumor suppressor function in normal cervical epithelium, liver, kidney, placenta, thymus, spleen, skeletal muscle and heart tissues.

Example 4: Construction and Transfection of Expression Vectors

An expression vector comprising the coding region of HCCS-4 gene was constructed as follows. First, the expression vector pCEV-LAC inserted with the HCCS-4 full-length cDNA clone obtained in Example 2 was digested with Sal I to obtain HCCS-4 full-length cDNA, which was then obtained from the eukaryotic expression vector N-terminal FLAG (Sigma, USA). Inserted into the Sal I site to obtain an expression vector N-terminal FLAG / HCCS-4. The expression vector was transfected with HeLa cervical cancer cell line (ATCC CCL-2) using lipofectamine (Gibco BRL) and then transfected by culture in RPMI medium containing 0.6 mg / ml G418 (Gibco). Cells were selected. At this time, HeLa cells transfected with the expression vector N-terminal FLAG containing no HCCS-4 cDNA were used as a control.

Example 5 Growth Curves of Cervical Cancer Cells Transfected with HCCS-4 Gene

To investigate the effect of HCCS-4 gene on the growth of cervical cancer cells, 1 x 10 ⁵ wild type HeLa cells, HeLa cervical cancer cells and vectors transfected with the vector N-terminal FLAG / HCCS-4 obtained in Example 4 HeLa cells transfected with N-terminal FLAG only were incubated for 9 days in RPMI medium, respectively. In each culture, cells adhered to the flask were trypsin (Sigma) and released, and the surviving cells were trypan blue dye exclusion, Freshney, IR, Culture of Animal Cells, 2nd Ed.AR Liss, New York (1987)) was counted on days 1, 3, 5, 7, and 9.

6 is a growth curve of wild type HeLa cells, HeLa cervical cancer cells transfected with the vector N-terminal FLAG / HCCS-4 obtained in Example 4 and HeLa cells transfected only with the vector N-terminal FLAG. As shown in FIG. 6, the mortality rate of HeLa cervical cancer cells transfected with vector N-terminal FLAG / HCCS-4 was higher than that of HeLa cells and wild type HeLa cells transfected with vector N-terminal FLAG. On day 9 after culture, only 50% of HeLa cervical cancer cells transfected with the vector N-terminal FLAG / HCCS-4 survived compared to wild-type HeLa cells. From these results, it can be seen that the HCCS-4 gene inhibits the growth of cervical cancer cells.

HCCS-4 gene of the present invention can be usefully used for the prevention and treatment of human cancer.

Claims (8)

Human cancer suppressor gene represented by SEQ ID NO: 1. The method of claim 1, And said cancer is a cancer of a tissue selected from the group consisting of normal cervix, liver, kidney, placenta, thymus, spleen, skeletal muscle and heart tissue. A human cancer suppressor protein encoded by the gene of claim 1 and having the amino acid sequence of SEQ ID NO: 2. 8. The method of claim 3, wherein And said cancer is cancer of tissue selected from the group consisting of cervix, liver, kidney, placenta, thymus, spleen, skeletal muscle and heart tissue. An expression vector comprising the gene of claim 1. A cell transformed with the vector of claim 5. The method of claim 6, Cells that are microorganisms or animal cells. The method of claim 7, wherein A cell that is Escherichia coli DH5α / HCCS-4 / pCEV-LAC (Accession No. KCTC 10178BP).