KR100378322B1 - Cancer cell-specific gene expression system - Google Patents

Abstract

The present invention relates to a cancer cell specific gene expression system, and more particularly to a cancer cell specific gene expression system comprising a promoter and a structural gene containing a binding site (E2Fbs) of a transcription factor E2F protein that is abnormally activated in cancer cells. It is about. Therefore, the gene expression system according to the present invention has no effect on normal cells, and can only provide a useful gene therapy method for cancer cells by only acting specifically on cancer cells with different structural genes.

Description

Cancer cell-specific gene expression system

The present invention relates to a cancer cell specific gene expression system, and more particularly to a cancer cell specific gene expression system comprising a promoter and a structural gene containing a binding site (E2Fbs) of a transcription factor E2F protein that is abnormally activated in cancer cells. It is about.

As molecular biology develops, the causes and progression of cancers are gradually revealed. Among them, mutations and abnormal expressions of oncogenes and tumor-suppressor genes are considered as the most important factors.

In the case of the retinoblastoma (referred to as RB) gene, which acts as a representative cancer suppressor gene with p53, abnormalities in the inactivation or gene structure / expression of this protein are found in about 30% of all carcinomas. [N. Engl. J. Med.,323, 1457-1462 (1990);N. Engl. J. Med.,330786-787 (1994);N. Engl. J. Med.,322, 1397-1398 (1990);N. Engl. J. Med.,330757-761 (1994).

In normal resting mammalian cells, RB proteins bind to E2F transcription factors to form an E2F-RB complex, which inhibits the transcriptional activation of E2F, thereby inhibiting the proliferation or cell cycle of mammalian cells. However, when cells receive the signals necessary for growth, RB proteins are phosphorylated by cell cycle-dependent kinase to dissociate the E2F-RB complex, thereby activating the E2F transcription factor. Enter the S group. At this time, E2F is a nucleic acid such as DNA polymerase α, thymidylate synthase, thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, and the like. It acts on the promoters of genes involved in cell cycle progression such as replication-related genes or cdc2, cdc6, B-myb, c-myb, c-myc, N-myc, cyclin A, cyclin D, cyclin E, cdk2, cdk4. Activates expression of Nucleic Acids Res. , 24 , 4139-4145 (1996); Cancer Biol ., 6 , 99-108 (1995); Science , 258 , 424-429 (1992); Gene , 237 , 281-302 (1999). Thus, the interaction between E2F-RB plays a pivotal role in regulating cell proliferation.

In addition, as a conventional invention using the characteristics of the transcription factor E2F as described above, US Pat. No. 5,885,833 discloses a base sequence in which a promoter contains an E2F binding site and a cell cycle gene homology region (CHR). Nucleic acid constructs are known which comprise as essential components. Since the nucleic acid constructs in the US patent show dependence on the cell cycle, there is a drawback in that it is not possible to induce constant expression for cancer treatment in vivo, as well as specific examples or embodiments of cancer cell specificity or activity strength of the nucleic acid constructs. No examples are given.

DNA cancer viruses, on the other hand, interfere with the normal interaction between E2F-RB to release the active form of E2F and thus activate the cell cycle, thus providing a major mechanism of cancer development.Nature,334, 124-129 (1988);Cell,54, 275-283 (1998).

By way of example, in cells infected with human papilloma virus (HPV), HPV E7 protein binds to RB family proteins (pRB, p107, p130, etc.), thereby inactivating RB function. Likewise, T antigens expressed by DNA cancer virus infections such as SV40 and adenoviruses, and carcinogenic proteins such as E1A also bind to RB proteins to inhibit the activity of intracellular RB proteins. The balance of the RB complex is broken and the expression of cytostatic genes is increased by the excess E2F released.

Such properties of the carcinogenic proteins of DNA cancer viruses eventually lead to uncontrolled cell proliferation, thereby leading to cell cancer.

Diseases caused by HPV infection include the most common cervical cancers, head and neck cancers, intraepithelial neoplasias and carcinoma of larynx, and juvenile flat warts. And the severity of each of these diseases varies from malignant to chronic.Curr. Opin. Oncol.,3, 191-199 (1999);Cancer Metastasis Rev., 15, 27-51 (1996);Int J. Dermatol.,27690-694 (1998);Am J. Surg. Pathol.,16, 641-649 (1992).

Recent studies have shown that in addition to the viruses mentioned above, Epstein-Barr virus (EBV) also acts by inhibiting the activity of RB proteins. It is known to bind to and inhibit the activity of RB protein. EBV is a herpesvirus that infects all races and causes Burkitt's lymphoma, lymphoproliferative syndrome, Hodgkin's disease, and nasopharyngeal carcinoma. [Leukemia,12, 1796-1805 (1998);Am J. Pathol.,155, 619-625 (1999);J. Virol.,73, 1630-1636 (1999);Indian j. Cancer,35, 47-56 (1998).

Therefore, in the case of treating cancer patients caused by the action of carcinogenic proteins as described above with gene therapy, normal cells and cancer cells can be distinguished and show pharmacological activity only in cancer cells without causing any side effects. Targeting is one of the most important challenges, and the need for a solution has been enormous.

Thus, the present inventors have made intensive research to solve the above problems, using the binding site of the E2F transcription factor that is specifically activated only in cancer cells to connect the therapeutic gene as a structural gene in the downstream. The present invention was completed by preparing an expression system and transfecting it into various cells to confirm that expression was specifically induced only in cancer cells.

Accordingly, a main object of the present invention is to provide a cancer cell specific gene expression system in which expression is selectively induced in E2F overactivated cancer cells such as cancer cells lacking the RB gene or cancer cells caused by DNA cancer virus infection.

1 shows a schematic diagram of plasmid (E2Fbs) _n / tk-LacZ according to Example 1 of the present invention.

2 shows a schematic diagram of plasmid (E2Fbs) ₆ / Sp1 / tk-LacZ according to Example 2 of the present invention.

Figure 3 shows a schematic diagram of the plasmid (E2Fbs) ₆ / Sp1 / tk-CD according to Example 3 of the present invention.

Figure 4 shows the change in the production of IL-2 from plasmid (E2Fbs) ₆ / Sp1 / tk-IL-2 and Sp1 / tk-IL-2 according to Example 4 of the present invention in COS-7 cells.

Figure 5 shows the change in production of TNF α from the plasmid (E2Fbs) ₆ / Sp1 / tk-TNF α and Sp1 / tk-TNF α according to Example 5 of the present invention in COS-7 cells.

The present invention relates to a cancer cell specific gene expression system comprising a promoter and a structural gene containing a binding site (E2Fbs) of the transcription factor E2F protein.

Referring to the present invention in more detail as follows.

The present invention relates to a cancer cell specific gene expression system comprising a promoter and a structural gene containing a binding site (E2Fbs) of a transcription factor E2F protein that is specifically activated only in cancer cells in which the RB protein group is inactivated. The use of the active type of free E2F in the activated cancer cells to increase the expression of cell proliferation genes, ultimately to replace the subjects whose expression is regulated by E2F from the cell proliferation gene to the therapeutic gene. In other words, if the therapeutic gene is bound to an E2F binding site (E2Fbs weakly referred to as 'E2Fbs'), the therapeutic gene will be selectively expressed only in specific cancer cells where E2F is abnormally activated and the cells proliferate. Cancer cells will die specifically in a short time. Accordingly, RB protein is phosphorylated by cell cycle-dependent kinase in cancer cells to dissociate the E2F-RB complex, thereby activating E2F transcription factors in cell survival or proliferation in normal cells. Instead of promoting the gene expression system of the present invention, activated E2F promotes the expression of therapeutic genes, rather than killing cancer cells. Such therapeutic genes include apoptotic genes, cytokine genes, immune function regulatory genes, antisense genes, tumor suppressor genes, and the like, including anticancer active gene systems combined with therapeutic genes having anticancer activity. Any conventional therapeutic gene known to have is applicable. In particular, the genes described above are more specifically described as cytosine deaminase, thymidine kinase, tumor necrosis factor α (TNF α), interleukin-2 (IL-2), and interleukin-12 ( IL-12), a combination of one or more of the genes encoding interleukin-18 (IL-18) or granulocyte / macrophage-colony stimulating factor (GM-CSF) and tumor suppressor gene p53 When inserted into the expression system of the present invention can be obtained an excellent anti-cancer activity effect.

The binding site (E2Fbs) of the transcription factor E2F protein of the gene expression system according to the present invention is a monomer or a multimer, and is preferably composed of 1 to 24 units.

In addition, the present invention can insert another transcription factor binding site with E2Fbs. As described above, when transfecting an cancer cell with an expression system in which E2Fbs and other transcription factor binding sites are inserted together, there is an advantage in that the expression ability is significantly increased compared to the expression system in which only E2Fbs is inserted. The reason why the expression ability is increased as described above is because the added transcription factor synergizes with E2F, unlike when expression is controlled only by E2F. Such transcription factor binding sites that can be inserted with E2Fbs include transcription factors Sp1, AP1, NF1 (ATF), or C / EBP binding sites. In addition, binding sites for those commonly known as transcription factors involved in gene expression. Can be inserted together.

In addition, the cancer cell-specific gene expression system of the present invention, as described above, the expression of foreign genes is very high and its persistence is also relatively high, and thus can be applied to the suppression treatment of all cancers in which E2F is specifically activated. These carcinomas include all carcinomas lacking the RB gene, such as retinoblastoma, and all carcinomas due to DNA cancer viral infections, particularly cervical cancer, head and neck cancer, intraepithelial neoplasia and laryngeal cancer. gene therapy for carcinoma of larynx, juvenile flat warts, Burkitt's lympoma, lymphoproliferative syndrome, Hodgkin's disease, and nasopharyngeal carcinoma useful.

More specifically, as an embodiment of the present invention as follows.

First, the characteristics of the cancer cells used in the present invention are as follows. COS-7 is a tumorous strain derived from SV40 infection of kidney green cells of African green monkeys.Cell,23, 175 to 182 (1981), expressing a T antigen; HeLa is a cell line derived from undifferentiated cervical carcinoma and expresses E7 [Cancer Res.,12, 264 (1952); 293 is a cell line derived from human embryonic kidney transformed with adenovirus and expresses E1A and E1B [J. Gen. Virol.,36, 59-72 (1977); Saos-2 is a cell line derived from osteogenic sarcoma, in which p53 and RB protein are not produced, but p107 or p130 similar to RB protein is normally expressed [J. Natl. Cancer Inst. (Bethesda),58, 209-214 (1997); CaSki is a human cell line derived from cervical epidermoid carcinoma that contains the HPV-16 type genome, which expresses E6 and E7.Science,196, 1456-1458 (1997); C3 is a mouse cell line transformed with human papilloma virus type 16, in which E6 and E7 are expressed [Eur. J. Immunol.,23, 2242-2249 (1993); NIH / 3T3 is a cell line derived from mouse fibroblasts and is a normal cell used as a control.J. Virol.,4, 549-553 (1969). The procedure for measuring the activity to confirm the expression of the gene expression system of the present invention in the above cancer cells is as follows. In other words, β-galactosidase (LacZ) gene of E. coli, which is easy to measure activity, is selected as a reporter gene, and herpes simplex virus (HSV) thymidine kinase (tk) is selected. The plasmid tk-LacZ is prepared to express the LacZ gene under minimal promoter control of the gene. Then, PCR was made of E2Fbs, which specific proteins can bind to E2F, inserted into plasmid pBS KS, and then (E2Fbs)_n(N = 2, 4, 6) is prepared. (E2Fbs) prepared above_nWas inserted into plasmid tk-LacZ to determine plasmid (E2Fbs)._n/ tk-LacZ was prepared, transfected with cancer cell lines COS-7, HeLa, 293, Saos-2, CaSki and C3 and the control group NIH / 3T3 by coprecipitation with calcium phosphate, and then cultured for 48 hours. It was. After incubation, the cultured cells were disrupted to measure β-galactosidase and luciferase activity, and the expression level of the reporter gene was calculated as a result. In this case, luciferase activity is used to correct the efficiency of transfection.

As a result, when plasmid (E2Fbs) n / tk-LacZ is transfected into E2F-activated cancer cells, E2F promotes the expression of LacZ gene, indicating that high β-galactosidase activity is detected in cancer cells. Can be.

Hereinafter, the process of preparing the E2Fbs of the present invention and various plasmids including the same and transfecting the prepared plasmids into cancer cells will be described in detail by the following examples, but the present invention is not limited thereto.

Example 1: Plasmids (E2Fbs) _n Manufacture of / tk-LacZ

(1) Preparation of the plasmid tk-LacZ

Plasmid pBlueLacZ (obtained from Dr. Hans Scholer of EMBL) was treated with restriction enzymes Bam HI, Klenow enzyme and restriction enzyme XhoI in order to isolate a 3.8 kb LacZ gene.

On the other hand, by simultaneously treating the restriction enzymes XhoI and SmaI to plasmid tk-CAT (obtained from Dr. Hans Scholer of EMBL), a plasmid containing the promoter of the tk gene was removed and the promoter of the tk gene was obtained. A plasmid combining the promoter of the tk gene and the isolated LacZ gene was prepared, and the plasmid was partially cut with the restriction enzyme Eco RI, and then inserted into a pBluscript cut with the same enzyme, whereby one Sp1 binding site and Plasmid tk-LacZ with only the TATA site was prepared.

(2) Preparation of plasmid pBS- (E2Fbs) _n

A plasmid containing an adenovirus E2A gene promoter (obtained from Dr. Dong-Soo Im, Biotechnology Research Institute) at 94 ° C using 20 ng, 50 pmol each of the E2-3 primers listed in SEQ ID NO: 1 and the E2-4 primers described in SEQ ID NO: 2 PCR (polymerase chain reaction) was performed by repeating 30 cycles of 1 minute at 63 ° C for 1 minute and 72 ° C for 45 seconds. The 93 bp fragment synthesized by PCR was treated with restriction enzymes XhoI and SalI to bind to each other to prepare E2F bound dimer (E2Fbs) ₂ , followed by plasmid pBS KS (Stratagen, USA). Plasmid pBS- (E2Fbs) ₂ was prepared by inserting at the XhoI / SalI position of the company. This plasmid by inserting the pBS- (E2Fbs) E2F dimer (dimer) obtained by process ₂ with restriction enzymes XhoI and SalI again in plasmid pBS- (E2Fbs) ₂ to prepare a plasmid pBS- (E2Fbs) _4.

The above procedure was repeated to prepare plasmid pBS- (E2Fbs) ₆ .

(3) Preparation of Plasmid (E2Fbs) _n / tk-LacZ

Each of plasmid pBS- (E2Fbs) ₂ , plasmid pBS- (E2Fbs) ₄ and plasmid pBS- (E2Fbs) ₆ was treated with restriction enzyme Eco RI and inserted 4.2 kb Eco RI DNA fragment of the tk-LacZ vector in the cut position. , Plasmid (E2Fbs) ₂ / tk-LacZ, plasmid (E2Fbs) ₄ / tk-LacZ and plasmid (E2Fbs) ₆ / tk-LacZ. Escherichia coli transfected with the plasmid (E2Fbs) ₆ / tk-LacZ prepared as described above was deposited on November 22, 1999 to the Institute of Biotechnology Gene Bank (KCTC) and assigned accession number KCTC 0698BP. received.

Example 2: Plasmids (E2Fbs) ₆ Manufacture of / Sp1 / tk-LacZ

Plasmid pBS- (E2Fbs) ₆ / Sp1 was prepared by cutting plasmid pBS- (E2Fbs) ₆ with restriction enzyme SalI and inserting a 0.2 kb SalI DNA fragment of plasmid tk-LacZ.

After the plasmid prepared above was treated with Eco RI, plasmid (E2Fbs) ₆ / Sp1 / tk-LacZ was prepared by inserting the 4.2 kb Eco RI DNA fragment used in Example 1. Escherichia coli transfected with the plasmid (E2Fbs) ₆ / Sp1 / tk-LacZ prepared as described above was deposited on March 16, 2000 to the Bank of Biotechnology (KCTC), accession number KCTC 0756BP Was given.

Example 3: Plasmids (E2Fbs) ₆ Manufacture of / Sp1 / tk-CD

Plasmid (E2Fbs) ₆ / Sp1 / tk-CD was prepared to investigate the properties and practical applicability of the plasmid prepared by binding the E2F transcription factor binding site and the therapeutic gene, cytosine deaminase (CD). It was. Plasmid pCD2 (obtained from Dr. Dong-Soo Lim) was cut with restriction enzymes Eco RI and Bam HI, treated with Klenow enzyme, and 1.5 kb DNA fragments were isolated.

Restriction of plasmid pGLN / SP73 / bGH, which includes the 3'- flanking sequence of rabbit β-globin (bGLN) introns and bovine growth hormone genes (bGH), for increased transcriptional efficiency and protein detoxification Simultaneously cut with enzymes Eco RI and Bam HI and treated with Klenow enzyme, the 1.5 kb CD fragment isolated above was inserted. This plasmid was digested with restriction enzyme NotI and treated with Klenow, and the plasmid pBS- (E2Fbs) ₆ / Sp1 prepared in Example 2 was treated with Xho I and Klenow enzyme and then separated. Plasmid (E2Fbs) ₆ / Sp1 / tk-CD was prepared by inserting a 0.8 kb (E2Fbs) ₆ / Sp1 / tk fragment.

Experimental Example 1: Determination of transcriptional activity by E2Fbs-containing plasmid in normal cells

10 μg of plasmid (E2Fbs) ₂ / tk-LacZ, plasmid (E2Fbs) ₄ / tk-LacZ, and plasmid (E2Fbs) ₆ / tk-LacZ, respectively, prepared in Example 1 and 1 μg of RSV-luciferase plasmid 2 x BBS (50 mM N, N-bis (2-hydroxyethyl) -22-aminoethanesulfonic acid (N, N-bis (2-hydroxyethyl) -22-aminoethanesulfonic acid), 280 mM NaCl and 1.5 mM Coprecipitation solution was prepared by adding 250 μl of Na ₂ HPO ₄ , pH 6.95) and 250 μl of 250 mM CaCl ₂ .

Plasmid HβA-LacZ was used as a positive control, and plasmid pBS was used as a negative control.

3 × 10 ⁵ cells of NIH / 3T3 cultured in DMEM medium with 10% fetal bovine serum and 0.45% glucose were dispensed in a 6 cm dish and incubated for 16 hours in a 5% CO ₂ incubator. The fresh medium was changed to 3 ml. After 4 hours coprecipitation solution was added and further incubated for 16 hours. The precipitate was washed with physiological saline dissolved in phosphate buffer, and 4 ml of fresh medium was added and incubated for 24 hours. The cultured cells were washed twice with phosphate buffer, 700 μl of phosphate buffer was dispersed, and the cells were collected in a 1.5 ml centrifuge tube using a rubber policeman and centrifuged at 4 ° C. and 4000 rpm for 4 minutes. It was. After centrifugation, the supernatant was removed and the cells were suspended by adding 50 [mu] l of dispersion (1 mM DTT in 250 mM Tris, pH 7.8). The cell suspension was frozen in liquid nitrogen and dissolved in a 37 ° C. thermostat three times to break up the cells, followed by centrifugation at 4 ° C. and 12000 rpm for 15 minutes to obtain cell extracts.

Luciferase activity was measured to correct the efficiency of transfection in the cell extract obtained by the above method. In addition, the relative activity of β-galactosidase expressed in normal cells NIH / 3T3 transformed by each plasmid was measured to determine the transcriptional activity by E2Fbs.

On the other hand, beta-galactosidase (luciferase) and luciferase activity measurement method used throughout the present invention is as follows.

1) Determination of activity of β-galactosidase

Add 40 μl of cell extract to 800 μl of solution 1 (60 mM Na ₂ HPO ₄ , 40 mM NaH ₂ PO ₄ , 10 mM KCl, 1 mM MgCl ₂ and 50 mM β-mercaptoethanol) on ice 200 μl of the solution (60 mM Na ₂ HPO ₄ , 40 mM NaH ₂ PO ₄ and ONPG 2 mg / ml) was added at second intervals and mixed vigorously, followed by reaction at 37 ° C. After the reaction, when color development occurred, the reaction was terminated by adding 500 μl of 1 mM Na ₂ CO ₃ at 30 second intervals, and the absorbance was measured at 420 nm.

2) Measurement of luciferase activity

To 350 μl of Solution A (2 mM ATP, 10 mM MgSO ₄ and 22.5 mM Gly-glycine, pH 7.8), 10 μl of the cell extract was added and solution B (D-luciferin 0.3 mg / Luminescence was measured using a luminometer (Lumat: EG & G Berthold) injecting 100 μl of ㎖ and 20 mM Gly-glycine (pH 7.8).

The measurement results are shown in Table 1 below.

As shown in Table 1, the expression of LacZ gene by E2Fbs was not observed in NIH / 3T3, which was normal cells, but only by the presence of binding sites such as Sp1, AP1, and NF1. The results suggest that RB protein binds to E2F in normal cells to inhibit the transcriptional activity of E2F.

Experimental Example 2: Transcriptional Activity by E2Fbs when Carcinogenic Protein E1A was Added

0.4 μg of an E1A-expressing plasmid (obtained from Dr. Dong-Soo Lim, Biotechnology Research Institute) was transformed into NIH / 3T3 cells in the same manner as the experiment of transcriptional activity in normal cells with RSV-luciferase plasmid and plasmid containing E2Fbs After the test, the relative activity of β-galactosidase in each cell culture was measured, and the results are shown in Table 2 below.

As shown in Table 2, the expression of the LacZ gene by E2Fbs was increased due to the expression of the carcinogenic protein E1A. Plasmid (E2Fbs) ₄ / tk-LacZ was about 30 times greater than plasmid tk-LacZ lacking E2Fbs, and plasmid (E2Fbs) ₆ / Sp1 / tk-LacZ was 9 times greater than plasmid Sp1 / tk-LacZ. The expression of the gene is increased. In the plasmids (E2Fbs) ₆ / AP1 / tk-LacZ and (E2Fbs) ₆ / NF1 / tk-LacZ, the expression of LacZ gene was increased by 9 times compared to plasmids AP1 / tk-LacZ and NF1 / tk-LacZ, respectively. It became. The above results are thought to be because E2F was activated by binding to E1A and RB proteins, leading to increased expression of LacZ gene.

Experimental Example 3: Transcriptional Activity by E2Fbs with Carcinogenic Protein E7

0.2 μg of an E7 expressing plasmid (obtained by Dr. Soo-jong Chun, Catholic University) was transformed into NIH / 3T3 cells in the same manner as the transcriptional activity test in the normal cells together with the plasmid containing RSV-luciferase plasmid and E2Fbs. Then, the relative activity of β-galactosidase in each cell culture was measured, and the results are shown in Table 3 below.

As shown in Table 3, the expression of E7 carcinogenic protein of human papilloma virus increased the transcriptional activity of the plasmid containing E2Fbs. In the case of plasmid (E2Fbs) ₄ / tk-LacZ, the LacZ gene was about 10-fold higher than plasmid tk-LacZ lacking E2Fbs, and plasmid (E2Fbs) ₆ / Sp1 / tk-LacZ was twice as large as plasmid Sp1 / tk-LacZ. Increased expression. In the case of plasmids (E2Fbs) ₆ / AP1 / tk-LacZ, (E2Fbs) ₆ / NF1 / tk-LacZ, LacZ gene expression was 2-3 times higher than that of plasmids AP1 / tk-LacZ and NF1 / tk-LacZ, respectively. This has been increased. This result is thought to be due to the activation of free E2F due to the inactivation of Rb protein by E7 induced the increased expression of LacZ gene.

Experimental Example 4: Transcriptional Activity by E2Fbs in SV40 T Gene Expressed Cells

The cell line was a COS-7 tumorous mutant expressing the T antigen, and the experiment was performed in the same manner as the transcriptional activity test in the normal cells except that the culture medium was replaced with DMEM-L medium containing 10% fetal bovine serum. The results are shown in Table 4 below.

As shown in Table 4, when plasmid (E2Fbs) ₆ / tk-LacZ was used, the expression of LacZ gene was increased by 51 times compared to plasmid tk-LacZ lacking E2Fbs, and the Sp1 binding site and (E2Fbs) ₆ In the case of plasmid (E2Fbs) ₆ / Sp1 / tk-LacZ, the expression of LacZ gene was increased by 123 times compared to the plasmid Sp1 / tk-LacZ. Plasmids (E2Fbs) ₆ / AP1 / tk-LacZ, (E2Fbs) ₆ / NF1 / tk-LacZ, and the like, were 100 and 124 times higher than the plasmids AP1 / tk-LacZ and NF1 / tk-LacZ, respectively. Expression was increased.

Experimental Example 5: Transcriptional Activity by E2Fbs in Cells Containing Low Dose Human Papilloma Virus Genome

The cell line was a HeLa tumor mutant expressing E7, and the culture was replaced with DMEM-L medium containing 10% fetal bovine serum, and the experiment was performed in the same manner as the transcriptional activity test in the normal cells. It is shown in Table 5 below.

As shown in Table 5, when the plasmid (E2Fbs) ₆ / tk-LacZ was used, the expression of LacZ gene was increased by 12-fold compared to the plasmid tk-LacZ lacking E2Fbs, and the plasmid containing the Sp1 binding site (E2Fbs). _{In the case of 6} / Sp1 / tk-LacZ, LacZ gene expression was increased by about 10-fold compared to the plasmid Sp1 / tk-LacZ. In the case of plasmids (E2Fbs) ₆ / AP1 / tk-LacZ, (E2Fbs) ₆ / NF1 / tk-LacZ, LacZ gene expression was about 10-11 fold higher than that of plasmids AP1 / tk-LacZ and NF1 / tk-LacZ, respectively. This has been increased.

Experimental Example 6: Transcriptional Activity by E2Fbs in Cells Expressing Adenovirus E1A Gene

Cell lines were transformed with adenovirus to express E1A 293 tumorous strains, except that the culture medium was replaced with MEM medium, the experiment was performed in the same manner as the transcriptional activity test in the normal cells, the results are shown in Table 6 Shown in

As shown in Table 6, when the plasmid (E2Fbs) ₆ / tk-LacZ was used, the expression of the LacZ gene was increased by 18 times compared to the plasmid tk-LacZ lacking E2Fbs, and the plasmid containing the Sp1 binding site (E2Fbs). _{In the case of 6} / Sp1 / tk-LacZ, the expression of LacZ gene was increased by 21 times compared to the plasmid Sp1 / tk-LacZ. Plasmid (E2Fbs) ₆ / AP1 / tk-LacZ and (E2Fbs) ₆ / NF1 / tk-LacZ also expressed LacZ genes by 23-24 fold compared to plasmids AP1 / tk-LacZ and NF1 / tk-LacZ, respectively. This has been increased.

Experimental Example 7: Transcriptional Activity by E2Fbs in Rb and p53 Gene Deficient Cells

The cell line was replaced with Saos-2, in which no Rb and p53 proteins were produced but normal expression of p107 or p130 similar to the RB protein, and the experiment was performed in the same manner as the transcriptional activity test in the normal cells. It is shown in Table 7 below.

As shown in Table 7, the expression of LacZ gene was increased in the case of using plasmid (E2Fbs) _n / tk-LacZ, compared to the plasmid tk-LacZ lacking E2Fbs, but the elevation was relatively higher than in the above examples. As low as. The results suggest that the expression efficiency of LacZ gene in plasmid (E2Fbs) _n / tk-LacZ does not increase because normally expressed RB protein p107 or p130 binds to E2F.

Experimental Example 8: Transcriptional Activity by E2Fbs in Cells Containing High HPV (Human Papilloma Virus) Gene

The cell line was a CaSki expressing E6 and E7 because the cell line contains the HPV-16 genome, and the culture medium was replaced with RPMI 1640 medium containing 10% fetal bovine serum. The experiment was performed as shown in Table 8 below.

As shown in Table 8, when the plasmid (E2Fbs) ₆ / tk-LacZ was used, the expression of the LacZ gene was increased by 475 times compared to the plasmid tk-LacZ lacking E2Fbs, and the plasmid containing the Sp1 binding site (E2Fbs) ₆ / Sp1 / tk-LacZ showed a 35-fold increase in LacZ gene expression compared to plasmid Sp1 / tk-LacZ.

Experimental Example 9: Transcriptional Activity by E2Fbs in Cells Artificially Introduced to Human Papilloma Virus Genome

The cell line was transformed into HPV-16 type C3 tumor cell line expressing E6 and E7, and the culture medium was replaced with IMDM medium including 8% fetal bovine serum and 1 × β-mercaptoethanol. Experiments were carried out in the same manner as the transcriptional activity test in cells, the results are shown in Table 9 below.

As shown in Table 9, in the case of plasmid (E2Fbs) ₆ / tk-LacZ, the expression of LacZ gene was increased by 4.8 times compared to the plasmid tk-LacZ lacking E2Fbs, and the plasmid containing the Sp1 binding site ( In the case of E2Fbs) ₆ / Sp1 / tk-LacZ, the expression of LacZ gene was increased by 11-fold compared to the plasmid Sp1 / tk-LacZ.

Experimental Example 10 Measurement of Cancer Cell Killing Capacity of E2Fbs Containing Plasmid Containing CD

Plasmid (E2Fbs) ₆ / Sp1 / tk-CD and plasmid Sp1 / tk-CD and the control plasmid pBS, which express the CD gene, which can be used for gene therapy prepared in Example 3, by expression of E7 C3 cells in which Rb protein is inactivated were transformed in the same manner as in the experiment of transcriptional activity in the normal cells.

Then, the culture of the transformed cells is replaced with a medium containing 5-FC (5-fluorocytosine) of 0, 0.5, 1, 5, 10, 50, 100, 500, 1000, 5000 and 10000 μM, Incubated for 3 days. The cultured cells were washed with phosphate buffer and incubated for 4 hours in a 5% CO ₂ incubator by adding 1 ml of MTT solution (5 mg / ml). After incubation, the supernatant was removed, mixed well by addition of 1 ml of DMSO, and the number of living cells was examined by measuring the absorbance at 590 nm.

Absorbance measurements yielded the relative IC ₅₀ of each plasmid in C3 cells (the dose at which 50% growth inhibition occurs; obtained from the logarithmic (dose-response) curve), and the results are shown in Table 10 below.

As shown in Table 10, the IC ₅₀ of the plasmid containing E2Fbs was reduced by 23 times compared to the plasmid without the E2F binding site. The results suggest that the RB protein activity was lost due to the expression of the E7 protein of HPV. The dissociation of E2F transcription factor significantly increased the activity of the tk promoter, resulting in high sensitivity to 5-FC.

Experimental Example 11: Safety Measurement in Normal Cells Using Rat Fetal Fibroblasts

According to the present invention, in the case of the vector in which the transcription factor Sp1 binding site is inserted together with E2Fbs, toxicity may be induced in normal cells by expression of a therapeutic gene resulting from Sp1 activity in normal cells. As a test cell, the activity of the gene expression vector inserted with the E2Fbs and Sp1 binding sites was measured.

The experiment was carried out in the same manner as in Experimental Example 1, except that the initial culture of fibroblasts isolated from the white rat fetus was used as normal cells for testing.

As shown in Table 11, not only the enhanced effect of E2Fbs transcriptional activity was detected in normal cells, but also E2Fbs inhibited the action of Sp1, thereby preventing unnecessary gene expression in normal cells and cancer cells of gene expression. It has been shown to further enhance specificity.

Example 4 Preparation of E2Fbs Containing Plasmids Containing IL-2 Gene, a Therapeutic Gene, and Measurement of IL-2 Production Capacity in Cancer Cells

Plasmid (E2Fbs) ₆ / Sp1 / tk-IL-2 and plasmid Sp1 / tk-IL-2 were prepared in the same manner by inserting cDNA of human IL-2 gene instead of CD gene as a therapeutic gene in Example 3. The plasmid prepared as described above was transformed into COS-7 cells in which RB protein was inactivated by expression of SV40 T antigen in the same manner as in the measurement of transcriptional activity in the normal cells of Experimental Example 1. After transfection of the culture of the transformed cells, 300 μl was recovered over 7 days from 24 hours every day and filled with the same amount of fresh medium each time.

In order to measure the amount of IL-2 present in the recovered culture, the biological activity of IL-2 was determined by MTT analysis using CTLL-2 cells, IL-2 dependent culture cells. That is, CTLL-2 cells were grown for 2 days in the medium to which the associated dilution of the recovered culture solution was added, washed with phosphate buffer solution, and 1 ml of MTT solution (5 mg / ml) was added to 4% in a 5% CO ₂ incubator. Incubated for hours. After incubation, the supernatant was removed, mixed well by adding 1 ml of DMSO, and the number of living cells was examined by measuring the absorbance at 590 nm.

Absorbance measurement results yielded the absolute IL-2 production of each plasmid in COS-7 cells, the results are shown in FIG. As shown in FIG. 4, COS-7 cells transfected with an E2Fbs-containing IL-2 expression vector produced continuously increasing amounts of IL-2 for the next 3 days, and the expression amount was up to 112,200 unit / ml. And gradually decreased from day 4. However, after 7 days, the expression of high levels of IL-2, reaching 24,980 units / ml, showed long-term expression of the therapeutic effect.

On the other hand, the amount of IL-2 expression of cells transfected with the Sp1 / tk-IL-2 vector without E2Fbs was minimal, in which case the difference with (E2Fbs) ₆ / Sp1 / tk-IL-2 was up to 180-fold. It was. This is because the gene expression from the (E2Fbs) ₆ / Sp1 / tk-IL-2 vector was inhibited by the SV40 T antigen binding to the RB protein and dissociating the RB-E2F complex in COS-7 cells. It is to prove that the enemy has been promoted. This led to the development of a new therapeutic gene expression system that selectively expresses IL-2 in RB protein-mutated cancer cells, and by using it, anti-cancer immunity by T cells, increased NK cell activity, and other induction of cytokine production. -2 anticancer treatment can be expected. The expression vector has a very high expression of foreign genes and a relatively high persistence, and thus, the expression vector is suitable for gene therapy for cancers related to DNA cancer virus infections such as cervical cancer.

Example 5: Preparation of E2Fbs containing plasmids bound to therapeutic gene TNF α and determination of IL-2 production capacity in cancer cells

Plasmid (E2Fbs) ₆ / Sp1 / tk-TNF α and plasmid Sp1 / tk-TNF α were prepared in the same manner as in Example 3 by inserting the human TNF α gene instead of the therapeutic gene CD in Example 3.

The plasmid prepared as described above was transformed into COS-7 cells in which RB protein was inactivated by expression of SV40 T antigen in the same manner as in the transcriptional activity test in the normal cells of Experimental Example 1. After transfection of the culture of the transformed cells, 300 μl was recovered over 7 days from 24 hours every day and filled with the same amount of fresh medium each time. In order to measure the amount of TNF α present in the recovered culture, the biological activity of TNF α was investigated by MTT analysis using Wehi 164 cells in which apoptosis was induced by TNF α. That is, Wehi 164 cells were grown for 2 days in the medium to which the associated dilution of the recovered culture solution was added, washed with phosphate buffer solution, and 1 ml of MTT solution (5 mg / ml) was added for 4 hours in a 5% CO ₂ incubator. Incubated for After incubation, the supernatant was removed, mixed well by adding 1 ml of DMSO, and the number of living cells was examined by measuring the absorbance at 590 nm.

Absorbance measurements resulted in the absolute production of TNF α of each plasmid in COS-7 cells, and the results are shown in FIG. 5. As shown in FIG. 5, COS-7 cells transfected with an E2Fbs-containing TNF α expression vector produced a continuously increasing amount of TNF α over the next 5 days, with expression amounts up to 16.15 μg / ml. On the 6th day, it gradually decreased. However, after 7 days, the production of TNF α decreased drastically due to the death of COS-7 cells themselves by TNF α. If there was no toxicity to the cell line for production by TNF α, it is expected that a significant amount of TNF α production will continue after 7 days as in the case of IL-2.

On the other hand, the amount of TNF α expression of cells transfected with the Sp1 / tk-TNF α vector without E2Fbs was minimal, in which case the difference with (E2Fbs) ₆ / Sp1 / tk-TNF α was up to 163,838 fold. This suggests that the gene expression from the (E2Fbs) ₆ / Sp1 / tk-TNF α vector was inhibited by the SV40 T antigen binding to the RB protein and dissociating the RB-E2F complex in COS-7 cells. To prove that it has been promoted. This has led to the development of a new therapeutic gene expression system that selectively expresses TNF α in cancer cells with RB protein mutations, and can be used to anticipate chemotherapy by TNF α. The expression vector has a very high expression of foreign genes and a relatively high persistence, and thus, the expression vector is suitable for gene therapy for cancers related to DNA cancer virus infections such as cervical cancer.

As described and demonstrated in detail above, the present invention relates to a cancer cell specific gene expression system comprising a promoter and a structural gene containing a binding site (E2Fbs) of the transcription factor E2F protein. Therefore, according to the present invention, unlike the normal cells, it was confirmed that the carcinogenic protein in cancer cells increases the transcriptional activity of E2F by interfering with the normal interaction between E2F and RB. By replacing the gene with a therapeutic gene in the proliferative gene, it was found that the therapeutic gene is expressed only in cancer cells with E2F activated, and the anticancer activity is very high. That is, as a result of transfection of the cytosine deaminase gene, which is a therapeutic gene having anticancer activity, as a cancer treatment gene, it was confirmed that this gene was expressed at high concentration in cancer cells and eventually killed. Experimental results showed an increase of 3.2 to 475-fold expression depending on the type of cancer cell line, especially when combined with (E2Fbs) ₆ . In addition, plasmids containing the interleukin-2 gene, tumor necrosis factor α, and other cancer treatment genes were transfected into cancer cells to trace gene expression. As a result, the vector containing (E2Fbs) ₆ contained no E2Fbs at all. The expression rate of the cancer treatment gene was up to 180-163,838 times higher than that of the non-vector. In addition, contrary to the original concern that by connecting the binding sites of other transcription factors such as E2Fbs and Sp1 to produce vectors inserted together and transfecting cancer cells, unwanted gene expression may occur in normal cells, Vectors with only Sp1 binding sites showed significant expression in normal cells, whereas vectors with both E2Fbs and Sp1 binding sites inserted E2Fbs inhibited the expression and thus enhanced cancer cell specificity of gene expression. Depending on the type of cancer cell line, the expression capacity was increased by about 2 to 11.5 times compared to the vector inserted only with E2Fbs. That is, a vector in which E2Fbs and other transcription factor binding sites are inserted together may be provided as an expression system having high expression ability and cancer specificity. Therefore, the gene expression system according to the present invention has no effect on normal cells, and has the effect of providing a useful gene therapy method for cancer cells by acting only on cancer cells by changing only the structural genes.

Claims (8)

Transcription factor E2F protein binding site (E2Fbs), Sp1, AP1, NF1 (ATF) and C / EBP transcription factor binding site is inserted, and the structural gene is included, so cancer cell expression is constantly independent of the cell cycle Cancer cell specific gene expression system, characterized in that possible. The cancer cell specific gene expression system of claim 1, wherein the structural gene is at least one selected from the group consisting of an apoptosis gene, a cytokine gene, an immune function regulatory gene, an antisense gene, and a tumor suppressor gene. The method of claim 1, wherein the structural gene is cytosine deaminase, thymidine kinase, tumor necrosis factor (TNF), interleukin-2, interleukin-12, interleukin-18, granulocyte macrophage A cancer cell specific gene expression system, characterized in that at least one selected from the group consisting of a gene encoding a colony stimulating factor (granulocyte / macrophage-colony stimulating factor; GM-CSF) and a tumor suppressor gene p53. According to claim 1, wherein the binding site (E2Fbs) of the transcription factor E2F protein cancer cell specific gene expression system, characterized in that the monomer (monomer) or multimer (multimer). The cancer cell specific gene expression system according to claim 1, wherein the binding site of the transcription factor E2F protein is 1 to 24. delete The cancer cell specific gene expression system according to any one of claims 1 to 5, wherein the cancer cell is a carcinoma cell that is deficient in RB gene or a cancer cell resulting from DNA cancer virus infection. 8. The method of claim 7, wherein the cancer cells are retinoblastoma, cervical cancer, head and neck cancer, intraepithelial neoplasias and carcinoma of larynx, juvenile flat warts, Burkitt's lymphoma. (Burkitt's lympoma), lymphoid proliferative syndrome (lymphoproliferative syndrome), Hodgkin's disease (Hodgkin's disease) and nasopharyngeal carcinoma characterized in that one of the cancer cell specific gene expression system.