JPH0514684B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to anticancer agents. More specifically, the present invention provides an anticancer agent containing an oligodeoxynucleotide or polydeoxynucleotide as an active ingredient, wherein the oligodeoxynucleotide or polydeoxynucleotide forms a hybrid with messenger RNA of an oncogene. The present invention relates to an anticancer agent characterized in that it has the same base sequence as a partial structure of a DNA sequence involved in hybrid formation in a DNA base sequence that can be used. Many cancers are caused by the abnormal expression of cancer-causing genes (oncogenes) built into the human body, and it is becoming clear that cancer would not develop if cancer-causing genes (oncogenes) were not present. The inventor first discovered messenger RNA of cancer-causing genes (oncogenes) that occur during the proliferation of cancer cells.
Based on this knowledge, we have obtained the knowledge that the proliferation of cancer cells is inhibited by supplying oligodeoxynucleotides or polydeoxynucleotides equivalent to the partial structure of a DNA sequence that can hybridize to the site where the messenger RNA is present. ,
Provided a method for inhibiting cancer cell proliferation (patent application 1982-
No. 87531). The present inventor further conducted repeated experiments in which the above-mentioned oligodeoxynucleotides or polydeoxynucleotides were applied to cancer-bearing animals, and as a result, succeeded in providing the anticancer agent according to the present invention. That is, the present invention provides an anticancer agent containing an oligodeoxynucleotide or polydeoxynucleotide as an active ingredient, as a means for carrying out the method for inhibiting cancer cell proliferation previously provided by the present inventor. A deoxynucleotide has a base sequence that is the same as a partial structure of a DNA sequence that is involved in hybridization among DNA base sequences that can hybridize to messenger RNA of cancer-causing genes (oncogenes). The present invention provides an anticancer agent characterized by the following. The present invention will be explained in detail below. The present inventor first used artificially synthesized RNA to directly add oligodeoxynucleotides equivalent to a partial structure of a DNA sequence that can hybridize to this RNA into a cell-free protein synthesis system. We discovered that the protein that should be synthesized based on this RNA is no longer synthesized (see Experimental Example 1 below). If you perform a similar experiment, this
It was confirmed that RNA-based protein synthesis takes place. Next, the present inventors discovered that α present in eukaryotic cells
Under physiological conditions using globin messenger RNA and β-globin messenger RNA,
Experiments were conducted using various types of DNA to determine whether they inhibit the production of α-globin and β-globin. (See Experimental Example 2 below) As a result, by adding DNA that can hybridize to α-globin messenger RNA to a direct, cell-free protein synthesis system, the production of α-globin is inhibited, and β-globin production is inhibited. It has been found that the production of β-globin can be inhibited by directly adding DNA that can hybridize to globin messenger RNA to a cell-free protein synthesis system. In addition, α-globin metsenjar
When using DNA that cannot hybridize to RNA, α-globin production is not inhibited, and when using DNA that cannot hybridize to β-globin messenger RNA,
It was also confirmed that the production of β-globin was not inhibited. Furthermore, in this experimental system, the
It has been found that when the DNA is an oligodeoxynucleotide, a high protein production inhibition rate is exhibited. Based on these experimental results, we confirmed that when SV40 DNA was administered to cells that had become cancerous due to the SV40 virus, the cancer cells were normalized. (See Experimental Example 3 and Experimental Example 4 below) Furthermore, the oncogenic gene SRC, which is generated during the proliferation of cells that have become cancerous due to the Rous sarcoma virus.
When the single-stranded DNA, which forms a double strand with the derived Messenger RNA, was administered to cells that had become cancerous due to the Rous sarcoma virus, it was confirmed that the growth of the cancer cells was significantly inhibited. (See Experimental Example 5 below) Furthermore, during the proliferation of Dowdy cancer cells in which expression of the oncogenic gene MYC has been confirmed, an oligodeoxy group equivalent to a partial structure of the DNA sequence that can hybridize to messenger RNA of the oncogenic gene MYC. It was confirmed that the proliferation of Dowdy cancer cells was significantly inhibited when nucleotides were administered (see Experimental Example 6 below).These experiments revealed that nucleotides can form hybrids to Messenger RNA derived from oncogenes. Although it has been revealed that directly adding DNA to cancer cells effectively inhibits cancer cell proliferation, the present inventors further discovered that
Based on this fact, we applied DNA that can hybridize to messenger RNA derived from oncogenes to tumor-bearing mice.
When conducting cancer treatment experiments, it was confirmed that the drug had a clear anticancer effect. The present invention has been made based on this knowledge. Since the anticancer agent of the present invention uses oligodeoxynucleotides or polydeoxynucleotides having a base sequence equivalent to a partial structure of a DNA sequence that can form a hybrid with messenger RNA derived from an oncogene,
It is a messenjar derived from other genes.
It does not hybridize with RNA and therefore
Use of the anticancer agent of the present invention does not affect protein synthesis by other genes. Currently, it is used as a cancer-causing gene (oncogene).
SRC, FPS, YES, ROS, MYB, ERB,
MYC, REL, MOS, RAS, BAS, ABL,
The existence of various genes such as FES, FMS, and SIS has been confirmed, but even for cancer-causing genes (oncogenes) that are currently unconfirmed, once they are confirmed, all cancer-causing genes (oncogenes) can be derived from that cancer-causing gene (oncogene). By using oligodeoxynucleotides or polydeoxynucleotides equivalent to substructures of DNA sequences capable of forming hybrids to messenger RNA of An anticancer agent can be provided. Hereinafter, oncogenic genes
Although the explanation will be given using Dowdy cancer cells, in which MYC expression has been confirmed, the anticancer agent of the present invention is not limited to this, but is applicable to cancers derived from all cancer-causing genes (oncogenes). be able to. Regarding the effects of oligodeoxynucleotides or polydeoxynucleotides used in the anticancer agent of the present invention on normal cells, the present inventors investigated the effect of oligodeoxynucleotides or polydeoxynucleotides used in the anticancer agent of the present invention on human normal cells using single-stranded DNA that can hybridize with messenger RNA derived from the SV40 virus. When the effects were investigated, it was found that the DNA does not affect the survival of normal cells, nor does it affect their ability to proliferate. Furthermore, during a therapeutic experiment on mice bearing Dowdy cancer cells, the behavior and appearance of the group treated with deoxynucleotides equivalent to the partial structure of the DNA sequence that can form hybrids against Messenger RNA derived from the oncogenic gene MYC was determined. No changes were observed. The oligodeoxynucleotides or polydeoxynucleotides used in the anticancer agent of the present invention are those obtained by degrading the DNA single strand of cancer-causing genes (oncogenes) by enzymatic treatment, or
It may be of any origin as long as it has a desired partial structure of a DNA sequence, such as one obtained by synthesizing a partial structure of a DNA sequence. Examples of methods for preparing oligodeoxynucleotides or polydeoxynucleotides used in the anticancer agent of the present invention include the following methods. One method is to clone a human cancer-causing gene (oncogene) and fragment a single strand of its DNA;
Another method is to clone oncogenes derived from RNA tumor viruses (retroviruses) that have a high degree of homology to human cancer genes (oncogenes), and fragment the single strands of that DNA. When cloning human cancer-causing genes (oncogenes), for example, λ phage or
Double-stranded cells using prosmid vectors such as pBR322
DNA, or use methods such as M13 phage to directly obtain single-stranded DNA. In order to replicate cloned DNA in large quantities, for example, phages containing oncogenes or E. coli carrying plasmids are grown in an appropriate volume of culture medium with antibiotics added as needed. Oncogenic genes (oncogenes) cloned after culturing and harvesting bacteria using conventional methods
Isolate the DNA containing the . In this separation,
DNA can be extracted by treatment with an organic solvent such as phenol or phenol-chloroform. To extract cancer-causing gene (oncogene) DNA from the isolated DNA, it is digested with restriction enzymes.
Use agarose gel electrophoresis, column chromatography, etc. The obtained cancer-causing genes (oncogenes)
DNA is fragmented as necessary. For this purpose, for example, treatment with a restriction enzyme such as endonuclease or sonication is performed. Fragment lengths typically range from 100 nucleotides to
A range of up to 9 nucleotide positions is considered. When preparing using M13 phage, a single-stranded fragment can be obtained directly, but considering the properties of the anticancer agent of the present invention, single-stranded DNA that can form a double strand with messenger RNA of an oncogene. must be selected and cloned. The oncogene DNA fragment obtained using pBR322 and λ phage is heated, for example, at 100° C. for 10 minutes and then rapidly cooled to make it a single strand. Since the product contains a single strand of the desired DNA fragment, it can be used as it is in the anticancer agent of the present invention. Since there are also single-chain fragments that are not complementary to , it is preferable to remove them using affinity chromatography or the like. Moreover, the oligodeoxynucleotide or polydeoxynucleotide used in the present invention can also be produced by organic synthesis. In this case, the base sequence of the oligodeoxynucleotide or polydeoxynucleotide is a messenger derived from an oncogene.
The base sequence must be capable of hybridizing with the RNA base sequence. For example, SRC
Gene uses positive DNA (5′-ATG, GGG,
AGC・AGC……) and minus DNA (3′−TAC・
CCC, TCG, TCG...) form a double strand, but among these, the TAC side (minus strand DNA) that forms a double strand with messenger RNA.
Select and synthesize DNA sequences. In addition, in the MYC gene, positive DNA (5′-ATG, CCC, CTC,
AAC...) and minus DNA (3′-TAC・GGG・
It forms a double chain of GAG/TTG...), but
Among these, the DNA sequence on the TAC side (minus strand DNA) that forms a double strand with messenger RNA is selected and synthesized. In this way, in the case of any oncogenic gene (oncogene), the messenger of the oncogenic gene (oncogene) that attempts to inhibit the proliferation of the oncogenic gene (oncogene)
Single strand that can hybridize to RNA
It is possible to select a base sequence of an appropriate length in a DNA base sequence, synthesize an oligodeoxynucleotide or polydeoxynucleotide of that base sequence, and use the oligo(or poly)deoxynucleotide. Next, the preparation of oligodeoxynucleotides or polydeoxynucleotides used in the anticancer agent of the present invention will be explained. Specific example 1 Creation of a vector containing an oncogene (oncogene) (1) Rous sarcoma virus (RSV) is applied to quail-derived cells (QT6) that have been made cancerous with a chemical substance, cultured, and RSV is isolated using Hirt's method.
- DNA was isolated. The cyclic covalently bonded chain obtained here (Co-valently closed circular,
CCC) DNA was cut with a restriction enzyme, Sac, and ligated with λgtwES/λDNA, which had been previously cut with a restriction enzyme, Sac, using an enzyme (ligase), and encapsulated in a protein of λ phage according to a conventional method. The completed λ phage was mixed with E. coli and allowed to form plaques on a Petri dish. Select those that hybridize with RSV-DNA from among the plaques, and extract RSV from this lambda phage using Sac.
− Excise the DNA and add Sal of pBR322 vector.
inserted in the position. Since the Sal position is located on the tetracycline resistance gene of the R plasmid, E. coli carrying this plasmid does not grow in the presence of tetracycline, but it was obtained by taking advantage of the fact that it can grow in the presence of ampirin.
A pBR322/RSV-DNA conjugate is obtained. (2) This pBR322/RSV-DNA conjugate contains all the genes of RSV, and was prepared using the method of Bishop et al. (De-lorbe et al., J.Vrol.36.50.1980).
If RSV-DNA is excised from lambda phage using another restriction enzyme instead of Sac in 1) above, it will contain only SRC-DNA.
A pBR322・RSV-SRC-DNA conjugate is obtained. Specific example 2 Production and purification of cancer-causing genes (oncogenes) (1) 2 containing ampicillin at a rate of 50 μg/ml
pBR322/RSV obtained in (1) of Specific Example 1 above for 1 ml of TSB (Tryptose Soy Broth) medium.
- Add 10 ⁶ to 10 ⁷ E. coli to the encapsulated DNA conjugate and culture with shaking at 37°C in a 20 ml test tube for 15 hours. This culture solution (E. coli per ml)
10 (containing ⁹ pieces) on a new medium of 2,
After culturing until the absorbance at 540 nm reached 0.5, 100 μg/ml of chloramphenicol was added, and the culture was further continued for 15 hours. The obtained E. coli suspension was centrifuged, and the obtained pellet was
% sucrose in 50mM Tris-HCl (PH8.0), 0.5ml of lysozyme solution (5mg/ml, manufactured by Worthington) and 1ml of EDTA (250mM).
mM, PH8.0). This was left at 0°C for 5 minutes, and 4 ml of Triton-X100 was added, and the resulting viscous liquid was centrifuged at 0°C and 30,000g for 30 minutes. Add 8 g of Cscl and 1 ml of ecidium bromide (5 mg/ml) to 8 ml of this supernatant,
Further centrifugation is performed at 100,000 g for 24 hours, the resulting crude plasmid fraction is collected, mixed with 2 times the amount of isopropyl alcohol, the upper layer is removed, and then dialyzed overnight with 0.1M Tris 10mM EDTA (PH8.0). . After concentrating the dialysed fluid and taking an amount equivalent to 50 μg of DNA, it was reacted with a restriction enzyme equivalent to 10 times the required amount, and then separated by agarose electrophoresis.
A fraction corresponding to RSV-DNA is extracted, concentrated, and dried using a conventional method to obtain double-stranded DNA containing cancer-causing genes (oncogenes). (2) pBR322・RSV− used in (1) above
Obtained in (2) of Example 1 instead of the DNA conjugate
A similar treatment using pBR322/RSV-SRC-DNA conjugate yields a double strand of the oncogenic gene (SRC). Specific example 3 Production of oncogenic gene fragment 5 μg of SV40 DNA (manufactured by Bethesde Research Laboratory) was mixed with 20 units of Hinf and heated at 37°C.
After reacting for 5 hours at 100°C, heat the DNA for 15 minutes to make the DNA into a single strand. The reaction mixture was adjusted to 137mM
NaCl, 5mM KCl, _{0.7mM Na2HPO4} , 6
mM dextrose and 21 mM HEPES were added and dissolved, and 2M CaCl ₂ was added so that the final concentration was 125 mM, and the resulting precipitate was collected after being left at room temperature for 20 to 30 minutes. This is used as is by adding it to the culture solution. Specific example 4 Using a DNA synthesizer (manufactured by Applied Biosystem), the minus DNA of MYC gene DNA,
Oligodeoxynucleotides (3'-...TAC, GGG, GCG,
20 deoxynucleotides (3′-GGG, TTG, CAA, TCG, AAG, TGG……−5′)
GAG・TTG・CAA・TCG・AAG・TG−5′)
was synthesized and purified. Specific example 5 Using a DNA synthesizer (manufactured by Applied Biosystem), the minus DNA of MYC gene DNA,
Of the oligodeoxynucleotides that form a double strand with positive DNA, 20 deoxynucleotides (3'-TGA, TGC, TGA, GCC, ACG, TCG,
GC-5′) was synthesized and purified. The anticancer agent of the present invention may contain the oligodeoxynucleotides or polydeoxynucleotides, as appropriate, in a suitable solvent ordinarily used in pharmaceutical formulations.
It is prepared as a solution, injection, suppository, etc. using excipients, adjuvants, etc. according to conventional methods for manufacturing pharmaceuticals. For formulation, desired oligodeoxynucleotides or polydeoxynucleotides may be used alone or in appropriate combinations, and other pharmaceutically active ingredients may be added. In these combinations and combinations, the ingredients to be combined or combined must not inhibit the anticancer effects of the oligodeoxynucleotides or polydeoxynucleotides mentioned above. . The above oligodeoxynucleotides or polydeoxynucleotides can be mixed with a suitable base that is inactive against them to form external preparations such as creams, ointments, and poultices. When preparing a solution or injection, distilled water for injection, physiological saline, aqueous dextrose solution, vegetable oil for injection, propylene glycol, polyethylene glycol, etc. can generally be used. Furthermore, if necessary, isotonizing agents, solubilizing agents,
Stabilizers, preservatives, soothing agents, etc. may also be added. Moreover, in the case of this type of dosage form, it is desirable to dissolve it in a sterile injection medium. Various bases and additives such as those mentioned above used in the preparation of formulations,
Needless to say, the adjuvant and the like must not inhibit the anticancer effect of the oligodeoxynucleotide or polydeoxynucleotide mentioned above. The anticancer agent of the present invention is applied directly to the cancer or administered intravascularly, so that the oligodeoxynucleotide or polydeoxynucleotide is adapted to the living body so that it can eventually reach the affected area. Examples of experiments to confirm the effects of the anticancer agent of the present invention using tumor-bearing mice are listed below. 5 μg of a mixture (1:2) of 20 deoxynucleotides obtained in Specific Example 4 and 20 deoxynucleotides obtained in Specific Example 5 and Dowdy cancer cells (1
×10 ⁸ cells/ml) was mixed with 0.1 ml and injected. From the next day, administer 0.1ml of TE buffer (10mM Tris-HCl (PH8.0), 1mM MEDTA-
5 μg of the above mixture (1:2) dissolved in 2Na) was injected into the site where the cancer cells were transplanted. In the control group, only TE buffer was injected at the same schedule as the drug administration group from the time of tumor injection. The formation and growth of subcutaneous tumors in both groups was observed for 50 days. When comparing tumor formation and tumor size with each group, tumor formation was observed in 4 out of 4 animals in the control group, whereas 3 in the drug administration group.
Tumors were not formed in two of the animals, and even in the one animal in which tumor formation was observed, the size of the tumor was smaller than that in the control group. From the above examples, it is clear that the anticancer agent of the present invention
It has been found to be extremely useful in suppressing the proliferation of cancer cells and preventing canceration in tumor-bearing animals. Examples of formulations of the anticancer agent of the present invention are listed below. Formulation Example 1 A substance similar to the 20 deoxynucleotide obtained in Example 4 was synthesized by the liquid phase method, and 2 g of the 20 deoxynucleotide was added to an isotonic MEM solution according to the usual method for manufacturing injections. 1 and sealed in an ampoule to prepare an injection. This injection contains 2 mg of the above 20 deoxynucleotide in 1 ml. Inject 1 to 5 ml of this injection at a time, depending on the symptoms, as close as possible to the site of tumor formation. Formulation example 2 RSV-SRC- obtained in specific example 2
2 g of DNA is added to an isotonic MEM solution and sealed in an ampoule to prepare an injection according to a conventional method for manufacturing injections. This injection contains 2mg of RSV-SRC-DNA in 1ml.
Contains. This injection is administered once or twice depending on the symptoms.
Inject 5 ml as close to the tumor site as possible. Next, the safety of the anticancer agent of the present invention will be explained by showing test examples. Test Example 1 20 deoxynucleotide obtained in Specific Example 4 and Specific Example 5 were administered to CBA/N nude mice.
When 250 μg/Kg of the 20 deoxynucleotide mixture (1:2) obtained in 1 was administered subcutaneously, no deaths or abnormalities were observed. As shown here, it is clear that the anticancer agent of the present invention is safe at the above-mentioned effective dosage. Examples of experiments conducted in connection with the present invention are listed below. Experimental Example 1 Inhibitory effect of polyadenylic acid and deoxypolyadenylic acid on polyphenyl-alanine synthesis reaction using polyuridylic acid as messenger RNA Polyuridylic acid (140 μg), ¹⁴ C-phenylalanine (10 ⁵ cpm, 300 μCi/μmole), Nieren- berg
and Mathaei's method (Proc.Nat.Acad.Sci.47
1588 (1961)) Escherichia coli extract, 13m
M magnesium acetate salt, 1mM ATP, 1mM
Creatine phosphate and creatine phosphokinase (1μg) were dissolved in 10mM Tris-HCl (PH7.5), an inhibitor was added to make a total volume of 40μ, and the mixture was reacted in a 1.5ml Etzpendorf tube at 37°C for 30 minutes. The amount of polyphenylalanine in the resulting solution was determined as the amount of radioprotein insoluble in 10% trichloroacetic acid at 95° C. using a liquid scintillation counter. The degree of inhibition was calculated by comparing the radioactivity of polyphenylalanine when the inhibitor was not added and the radioactivity when the inhibitor was added. The results are shown in Table 1.

[Table] As shown in Table 1, polyuridylic acid (RNA)
On the other hand, polydeoxyadenylic acid (DNA) corresponding to the base sequence showed a remarkable activity of inhibiting the synthesis of polyphenylalanine (protein). Also,
DNA strands showed strong inhibitory activity up to oligodeoxyadenylates (oligonucleotides) of 9 or more nucleotides. Experimental Example 2 Specific inhibition effect of globin single-stranded DNA on globin synthesis in the reticulocyte system of rabbits.
This is an example of an experiment in which it was confirmed that protein synthesis can be inhibited under physiological conditions using oligodeoxynucleotides or polydeoxynucleotides that hybridize to RNA. 4.2mM calcium phosphate, 2mM dimethylthiothreitol (DTT), 0.08mM amino acids (excluding methionine), 6mM potassium acetate, 8mM
Magnesium acetate, 4.5 μg spermidine, ³⁵ S-methionine (0.1 μCi), and 10 μg of a cell-free reticulocyte protein synthesis system prepared by the method of Jackson and Pelham et al. (Eur. J. Biochem. 67.247-256 (1976)).
, α and β globin messenger RNA each
Dissolve 75μg in 21mM HEPES and add the inhibitors listed in Table 2 to make a total volume of 30μ, then add 1.5ml.
The reaction was carried out in an Etzpendorf tube at 30°C for 30 minutes. The reaction products in the solution obtained by the above reaction were separated into α globin, β
The protein was separated into globin and other proteins, and the radioactivity of each fraction was measured using radioautography. The degree of inhibition was calculated by comparing with the radioactivity when no inhibitor was added. The results are shown in Table 2.

[Table] As shown in Table 2, the genome of α-globin
DNA specifically inhibits only the synthesis of α-globin, and genomic DNA of β-globin specifically inhibits only the synthesis of β-globin. Calf thymus DNA showed almost no inhibitory activity. This indicates that the presence of DNA corresponding to the base sequence of messenger RNA in eukaryotic cells specifically inhibits protein synthesis based on the messenger RNA. moreover,
Since the 15-oligodeoxynucleotide corresponding to the N-terminal amino acid of α-globin inhibits only the synthesis of α-globin, and the 15-oligodeoxynucleotide of M13phage has little effect on the synthesis of α-globin and β-globin, it was found that It was found that even when the DNA strand corresponding to the RNA base sequence was shortened to 15 nucleotides, its inhibitory activity and specificity were not lost. Experimental Example 3 Inhibitory effect of SV40 single-stranded DNA on the growth of SV40-infected mouse cancer cells (1) SV40-infected cancer cell growth Microplate (manufactured by Linbro, hole inner diameter
1.5 cm) were filled with 6 x ¹⁰⁴ mouse 3T3 cells that had become cancerous due to SV40 infection per well.
Suspend in DULBECCO-EAGLE culture medium (containing 10% bovine serum protein) and culture in 5% carbon dioxide gas for 24 hours. After removing the culture solution, a solution of DNA (50mM HEPESPH
7.1, 270mM NaCl, 1.5mM _NaHPO4 .
Add 26μ of 12H ₂ O), add 302μ of freshly prepared MEM culture solution (containing 10% bovine serum protein), and culture in 5% carbon dioxide at 37°C for 12 hours. After culturing, the cells are detached from the microplate by trypsin treatment, some are cultured on soft agar medium for one week according to the standard method, and some are transferred to a culture dish and their morphology and degree of proliferation are observed under a microscope every day. did. (2) Effect determination After culturing on a soft agar medium, the number of colonies in the soft agar medium was counted and compared with the control to determine the degree of inhibition. The results are shown in Table 3.

[Table] As shown in Table 3, SV40 DNA significantly inhibited the proliferation of cancerous cells, but salmon spermatozoa DNA and
RSV-DNA showed no inhibitory activity. Furthermore, in this experiment, the morphology of cancer cells changed to that of normal cells when observed under a microscope, and normalization of cancer cells was also confirmed morphologically. Experimental Example 4 Inhibitory effect of SV40 single-stranded DNA on growth of SV40-infected human cancer cells (1) Growth of SV40-infected cancer cells Microplate (manufactured by Linbro, hole inner diameter
1.5cm) were filled with 6 x ¹⁰⁴ human IMR90 cells that had become cancerous due to SV40 infection per well.
Suspend in DULBECCO-EAGLE culture medium (containing 10% bovine serum protein) and culture in 5% carbon dioxide gas for 24 hours. After removing the culture solution, a solution of SV40 DNA (50mM HEPES
PH7.1, 270mM NaCl, 1.5mM _NaHPO4 .
Add 26μ of 12H ₂ O), add 302μ of freshly prepared MEM culture solution (containing 10% bovine serum protein), and culture in 5% carbon dioxide at 37°C for 12 hours. After culturing, the cells were detached from the microplate by trypsin treatment, transferred to a culture dish, and their morphology and degree of proliferation were observed under a microscope every day. (2) Effect judgment In observation under a microscope, SV40DNA0.08μ
In the group treated with g/6×10 ⁴ cells, the number of cancer cells was significantly lower than in the untreated group, and the morphology of the remaining cancer cells became that of normal cells, and the cancer cells also normalized morphologically. The growth inhibitory effect on human cancer cells was confirmed. Human ^normal cells were also tested in the same way.
Even when human normal cells were treated with 12.5 times more, there was no change compared to the untreated group, and no effects on human normal cells were observed. Experimental example 5 Inhibitory effect of RSV-SRC single-stranded DNA on proliferation of RSV-infected cancer cells (1) Microplate (manufactured by Linbro, hole inner diameter
Place 6 x ¹⁰⁴ avian fibroblasts that have become cancerous due to RSV infection per hole in a 2.5 cm), suspend them in F10 culture medium (containing 10% bovine serum protein), and culture in 5% carbon dioxide gas for 24 hours. do. Remove the culture medium, add new F10 culture medium (containing 10% bovine serum protein) containing 26 μ of DNA (complex with calcium phosphate) as an inhibitor, and incubate at 37°C in 5% carbon dioxide gas.
Incubate for 12 hours. After culturing, the cells were detached from the microplate by trypsin treatment and cultured in soft agar medium for one week according to a conventional method. (2) Effect determination After culturing in soft agar medium, the number of colonies in the soft agar medium was counted and compared with the control to determine the degree of inhibition. The results are shown in Table 4.

[Table] As shown in Table 4, RSV-SRC-DNA significantly inhibited the proliferation of cancerous cells, but
DNA and SV40DNA showed no inhibitory activity. RSV-SRC-DNA is a type of cancer-causing gene (oncogene). Due to this,
Corresponds to the base sequence of messenjar RNA derived from oncogenes (oncogenes) in cancer cells
It can be seen that only single-stranded DNA specifically suppresses the proliferation of cancer cells. Experimental Example 6 Effect of oligodeoxynucleotides on proliferation of Dowdy cultured cancer cells The following experiment confirmed that oligodeoxynucleotides capable of forming hybrids with oncogenes significantly inhibited the proliferation of cancer cells in an in vitro test. . Dowdy cancer cells with 10% fetal bovine serum
1x in RPMI1640 culture solution (manufactured by Gibco)
The cells were suspended at a concentration of 10 ⁴ cells/ml, and 0.1 ml of this was spread onto a 96-well microplate (manufactured by Coaster), and allowed to stand in a carbon dioxide gas incubator for 24 hours. Thereafter, the 20 deoxynucleotide obtained in Example 4 was dissolved in RPMI1640 culture solution, and 100μ
5μ of this was taken and added to each well, and from the next day, the same procedure for adding 20 deoxynucleotides was continued once a day for 6 consecutive days. In the control experiment, only RPMI1640 culture solution was added in the same manner. The number of living cells in each well was counted every day by trypan blue staining. As shown in Figure 1, only when the above 20 deoxynucleotides were added, cell proliferation stopped on the 3rd day, after which the cell number began to decrease, and on the 6th day, 1×
It became less than 10 ⁴ cells.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the test results of Experimental Example 6 described herein. The vertical axis of the graph represents the number of cancer cells/ml, and the horizontal axis represents the number of days of culture.
-●- indicates a drug-free group, and -Γ- indicates a drug-administered group.

Claims (1)

[Claims] 1 An anticancer drug containing oligodeoxynucleotides or polydeoxynucleotides as an active ingredient, in which the oligodeoxynucleotides or polydeoxynucleotides are capable of hybridizing to messenger RNA of cancer-causing genes (oncogenes). An anticancer agent characterized by having a base sequence identical to a partial structure of a DNA sequence involved in hybrid formation.