SUBSTANCE: therapy involves introduction of sonicated human DNA combined with nonhistone protein to the patient with the length of human DNA fragments making 200-6000 pairs of bases, and the ratio of sonicated human DNA/nonhistone protein being 1/0.8. The complex is administered in a dose to ensure the blood concentration of sonicated human DNA 25 ng/ml - 1000 ng/ml.
EFFECT: application of the invention implies oncotherapy ensured by substitution of replacement oncolocus by a DNA fragment with nonmutant allele to form a nucleotide stable complex.
2 cl, 4 tbl, 11 dwg
The invention relates to medicine and can be used in the treatment of patients with drugs fragmented human DNA in injection form or in the form of a microenema in nucleotides stable complex with gestonorone protein Protamine used as leucotonalite in the treatment of cancer and as an anti-cancer agent.
There is a method of treating patients using the drug with antitumor, antioxidant and radioprotective action, representing fragmented to 200-2000 nucleotides nucleoprotein complex content, wt.%: double helix DNA 60-80 and proteins of the nuclear matrix 20-40, upon receipt of which the use of species-specific biological raw materials - the human placenta [EN 2234323, AK 48/00, 2004, 27.05.2003. The drug with antitumor, antioxidant and radioprotective action].
The disadvantage of this method is the relatively low efficiency of the treatment.
The closest in essence to the present invention is a method of treating cancer based on the introduction into the patient fragmented DNA, which is used homologous DNA having a biologically active amount of the complete genome physiologically and genetically healthy donor, the ri this DNA is injected dose, creating such a DNA concentration in the blood plasma, which is equal to or more than the concentration of the DNA in the blood plasma, but does not exceed the maximum permissible concentration equal to 30 μg/ml [EN 2313349, A61K 48/00, 2006, 16.01.2006. The method of treatment of cancer].
In this way the fragmented DNA is not protected from the action of nucleases human blood and cells are delivered primarily short DNA fragments. To deliver larger fragments of exogenous DNA using the maximum dose of fragmented DNA (closer to 1500 ng/ml). Only in this case, the cell delivered a number of full-sized DNA fragments, which are able to repairbot the damage zone associated with the loss of a single nucleotide or a fragment of chromosomal DNA.
This shows the lack of effectiveness of treatment, because even at the maximum possible concentrations of therapeutic DNA in the blood equal to 1500 ng/ml, not all cells where there has been a loss of genetic material, enough has kept its full-size fragments of exogenous DNA.
All this reduces the effectiveness of the treatment of leukopenia caused by the use of cytotoxic drugs, and the effectiveness of treatment of patients with oncological diseases, as active principle - fragmented DNA undergoes immediate nucleotides degradation by nucleases in the blood plasma, and to achieve therapeutic effect it is necessary to apply the maximum concentration of the drug.
The desired result is to increase the effectiveness of treatment patients receiving software chemotherapy, and patients with cancer.
The desired result is achieved that the method of treatment of patients with leukopenia caused by software chemotherapy, and patients with cancer, based on the introduction into the body fragmented human DNA, fragmented human DNA is administered in combination with nonhistone protein Protamine.
In addition, the desired result is achieved by the introduction into the body of fragmented human DNA in complex with nonhistone protein Protamine produce parenteral or microcosmographie.
In addition, the desired result is achieved by the fact that the ratio of fragmented DNA/nonhistone protein Protamine is 1/0,8.
In addition, the desired result is achieved by the fact that the introduction of fragmented human DNA in complex with nonhistone protein Protamine produce at a concentration of fragmented human DNA, equal at 25 ÷ 1000 ng/ml.
The impact of fragmented human DNA in complex with nonhistone protein Protamine at a concentration of 25 ng/ml oznacza is t, that is 1000 times less than the maximum allowable concentration equal to 30 μg/ml, and 10 times less than the concentration of fragmented DNA that is not associated with Protamine, which first appears the effect.
More broadly, the present invention is to develop experimentally confirmed way joint effects of the drug fragmented human DNA and nonhistone protein Protamine in the treatment of patients with leukopenia caused by chemotherapy used in the treatment of cancer, and patients with cancer, when fragments of therapeutic DNA is protected by a complex of Protamine from the action of nucleases blood. Stabilization of the fragments of the fragmented human DNA, which can be used the drug, "Anagen", nonhistone protein Protamine enhances its therapeutic opportunities, reflected in the fact that when carrying out homologous recombination, capture more extended regions of the genome, which increases the probability of falling into the area of the substitution mutations that led to a crash in proliferative activity netransformovany cells or defects in the genome of the CCM formed as a result of heavy chemotherapy or gamma irradiation.
In modern literature there are no indications of pre the proposed method for the treatment of patients with leukopenia, caused by software chemotherapy, and patients with cancer, which uses complex fragmented human DNA with nonhistone protein Protamine.
Therefore, the proposal meets the criteria of novelty and inventive step.
Listed below are theoretical and experimental data, confirming that the invention meets the criteria practical (industrial) applicability.
The drawings show:
figure 1 - electrophoretic characterization of exogenous therapeutic DNA human placenta used for injection into the body experienced mice, where 1 is the molecular weight marker BssT1I hydrolyzate of phage λ, the digits to the left of the block marked fragments of the appropriate molecular weight in TPN;
figure 2 - nucleara activity of the blood serum of the mouse, where M is the marker of molecular weights, numbers indicate the sizes of the corresponding fragments in TPN; 2 - the original human DNA fragmented by hydrodynamic method to fragments ranging in size from 200 to >6000 BP; 3 - human DNA, incubated with 1% serum mouse, 4 with 10% serum of the mouse 5 - 50% 6 - 97% serum mouse;
figure 3 - protection of DNA associated with different (by weight) the amount of Protamine, from the action of nucleases during incubation in 10% serum of the mouse, where (a) - Hind III hydrolysis of the DNA of phage λ, b) - fragmented human DNA. On top of the gel indicate the content of Protamine in the sector in relation to DNA in %. Left shows the fragments of the appropriate molecular weight in TPN;
figure 4 - comparison leucotonalite activity of exogenous human DNA (8) and human DNA associated with Protamine (9), injected into mice after exposure of the cytostatic cyclophosphamide; 7 - control group of mice, without affecting DNA;
figure 5 - number of labeled material (9 - DNA associated with Protamine, or 8 - unprotected DNA)in tumors at different times after injection;
figure 6-a) electrophoregram labeled λ Hind III DNA after intravenous administration to mice and allocation microsomes fraction of tumor cells in mice solid (10) and ascitic (11) form; b) electrophoregram labeled λ Hind III DNA-free (12, 13) and is associated with Protamine (14, 15), isolated from the blood of mice after 5 min (12 and 14) and 20 min (13 and 15) after its intravenous administration. M - molecular weight marker, Hind III digest DNA of phage λ, the numbers indicate the fragments of the appropriate molecular weight in TPN DNA is delivered into the tumor in a degraded form. Unprotected DNA in 5 min degrades under the influence of nucleases blood. DNA associated with Protamine, is present in the blood after 20 min in the high-molecular form;
figure 7 - the step on the growth of experimental tumors mouse exogenous DNA in an unprotected form (8) and associated with Protamine (9);
on Fig morphological differences fragmented exogenous DNA delivered in micromason space cells MCF-7 in complex with Protamine (b) and without Protamine (a). Left - electrophoretic characteristics in a 0.7% agarose gel exogenous DNA found in mikromazernom kernel space of MCF-7 cells after culturing in a medium containing exogenous DNA concentrations: for unprotected DNA, 100 μg/ml, protected Protamine DNA 25 µg/ml on the Right - radiograph agarose block after drying. The numbers on the blocks indicate the time of incubation (min) culture of cells labeled with α32R extracellular human DNA and α-P32dATP (180α); digits to the left of the block marked fragments of the appropriate molecular weight in TPN;
figure 9 - evolution of the delivery of fragments of exogenous DNA associated with Protamine (9) and unprotected Protamine (8), in the cytoplasm (a) and micromason space (b) nuclei of MCF-7 cells. Dynamics of appearance of labeled material in a fraction of the chromatin of the nuclei of MCF-7 cells (C);
figure 10 - analysis of apoptosis induction of cell adenocarcinoma human breast MCF-7, incubated in the presence of 100 μg/ml unprotected Protamine fragmented DNA of the human placenta (18), 25 μg/ml human DNA associated with Protamine (19), in the absence of DNA (17). Culture (17, 18, 19) in what was aziraphale apoptosis TNF-α at a concentration of 100 µg/ml for 24 hours in an environment containing 10 µg/ml cycloheximide. (16) DNA isolated from untreated MCF-7 cells. M - molecular weight marker, numbers to the left of the block marked fragments of the appropriate molecular weight in the gel In both cases (18 and 19) in the presence in the cultural environment of exogenous DNA induced apoptosis and formed a ladder of DNA fragments that characterize apoptotic degradation of nuclear chromatin;
figure 11 - analysis of apoptosis induction of cell adenocarcinoma human breast MCF-7, incubated in the presence of unprotected Protamine fragmented DNA in the human placenta at concentrations of 25 μg/ml (20), 2.5 µg/ml (21), 0.25 µg/ml (22) and 0.025 µg/ml (23) and human DNA associated with Protamine at concentrations of 25 μg/ml (19), 2.5 µg/ml (24), 0.25 µg/ml (25) and 0.025 µg/ml (26); the digits to the left from block marked fragments of the appropriate molecular weight in gel
The proposed method for the treatment of patients with leukopenia caused by software chemotherapies in the treatment of cancer, (leucotonalite tool) and patients with cancer (an anti-cancer agent) is implemented as follows.
The drug fragmented human DNA, used as monotherapy as leucotonalite and as an anti-cancer agent in injection form or in the form microcl the sm, mixed immediately before introduction into the body with a solution of nonhistone protein Protamine in a ratio of 1 part DNA / 0.8 parts of Protamine for forming nucleotides stable complex.
The effective concentration of DNA protected by Protamine complex can be significantly lower than fragmented DNA in an unprotected form (minimum of 4 times), and leucotonalite and anticancer activity of the drug fragmented human DNA remains unchanged.
In addition, stabilization of DNA fragments (drug, "Anagen") nonhistone protein Protamine expand its therapeutic opportunities, reflected in the fact that when carrying out homologous recombination, capture more extended regions of the genome, which increases the probability of falling into the area of the substitution mutations that led to a crash in proliferative activity netransformovany cells or defects in the genome of the CCM formed as a result of heavy chemotherapy or gamma irradiation.
An example implementation of the method.
The drug fragmented DNA used as leucotonalite or anti-cancer agent, is injected into the patient by injection or microenemas 2-6 times per day to 5 mg of active substance (fragmented human DNA). Just before doing one is Lacona mixed drug fragmented DNA 5 mg (in solution) and 4 mg of protein Protamine (in solution). Nucleotides stable complex is formed immediately (a few seconds) at room temperature. After mixing and formation of the complex of the drug is injected into the patient.
To confirm that the proposed method can achieve the desired result, will bring his theoretical and experimental validation.
In our earlier work [Yakubov LA, Popova NA, Nikolin VP, Semenov DV, Bogachev SS, Os'kina IN. Increasing interest among genomic DNA protects mice against radiation and chemical mutagens. Genome Biol. 2003; 5: 3; Khegai II, Bogachev SS, Os'kina IN, Popova NA, Semenov DV, Shurdov MA, Yakubov LA. Changes in the symptoms of hypothalamic diabetes insipidus after treatment with homologous exogenous DNA. Dokl Biol Sci. 2004 May-Jun; 396: 233-235; Nikolin V.P., Popov N.A., Sebelia IE, Strunkin D. N., Rogachev V.A., Semenov D.V., S. Bogachev, Yakubov L.A., Shutov M.A. the Effect of exogenous DNA on the growth of experimental tumors. Oncology issues. 2006. T, No. 1, p.66-69; Nikolin V.P., Popov N.A., Sebelia IE, Strunkin D. N., Rogachev V.A., Semenov D.V., S. Bogachev, Yakubov L.A., Shutov M.A. the Effect of exogenous DNA on the restoration of leucopoiesis and antitumor effect of cyclophosphamide. Oncology issues. 2006. T, No. 3, s-340; Rogachev VA, Likhacheva A, Vratskikh O Mechetina LV, Sebeleva THOSE Bogachev SS, Yakubov LA, Shurdov MA. Qualitative and quantitative characteristics of the increasing interest among DNA delivered to the nucleus of a living cell. Cancer Cell Int. 2006 Oct 11; 6:23; Likhacheva AS, Nikolin VP, Popova NA, Dubatolova TD, Strunkin DN, Rogachev VA, Sebeleva THOSE Erofeev IS, Bogachev SS, Yakubov LA, Shurdov MA. Integration of human DNA fragments into the cell genomes of certain tissues fromadult mice treated with cytostatic cyclophosphamide in combination with human DNA. Gene Ther Mol Biol. 2007; 11: 185-202; Likhacheva AS, Nikolin VP, Popova NA, Rogachev VA, Prokhorovich MA, Sebeleva THOSE Bogachev SS, Shurdov MA. Exogenous DNA can be captured by stem cells and be involved in their rescue from death after lethal-dose γ-radiation. Gene Ther Mol Biol. 2007; 11: 305-314; Yakubov LA, Rogachev VA, Likhacheva AC, Bogachev SS, Sebeleva THOSE Shilov AG, Baiborodin SI, Petrova NA, Mechetina LV, Shurdov MA, Wickstrom E. Natural human gene correction by increasing interest among small genomic DNA fragments. The Cell Cycle. 2007 Jul; 6(18): 2293-2301] we have described pleiotropic effects of fragmented exogenous DNA in the living cell. There were separate groups differ in mechanism of the types of impacts that could occur as the following describes the phenomenon.
1. Leucotonalite when mielosupression caused by cytostatic drug cyclophosphamide.
2. Radioprotectors and radiotherapy effect.
3. Inhibitory effect on the growth of experimental tumors in mice.
4. Participation in reparations mutant allele of caspase 3 cells adenocarcinoma human breast MCF-7.
5. The ability to integrate into the recipient genome (fragments of human DNA integrates into the genome of the mouse) during repair miaocheng crosslinks caused by the action of the cytostatic cyclophosphamide.
In the experiments performed in the analysis of these phenomena, we used a large number of exogenous DNA that have ensured its participation in the molecular processes governing these phenomena.
In the present work we use is ovali histomorphology protein Protamine to protect fragmented exogenous DNA from the action of nucleases in the blood plasma and culture medium during growth of the cell culture. As follows from the experimental results, the ability fragmented exogenous DNA associated with Protamine, to show leucotonalite action to inhibit the growth of experimental tumors of the mouse and to participate in the reparations mutant allele of caspase 3 cells adenocarcinoma human breast MCF-7 is maintained. Fragments of exogenous DNA that is protected by Protamine, not degrade in the body and in the culture medium. This allows you to use a significantly smaller amount of exogenous DNA to determine these types of its pleiotropic actions.
The methodology used.
Studies were conducted on mice-males line IAS wiring of the vivarium of the Institute of Cytology and genetics SB RAS. Mice were kept in plastic cages of 10 animals each with free access to food and water.
Preparations of human DNA was obtained from placentas of healthy parturients. For DNA extraction used butfinally method that allows to obtain the full genome, maintaining the DNA fragments, which in vivo is strongly associated with the nuclear matrix proteins (scaffold). Fragmentation of DNA was carried out by ultrasonic disintegrator at a frequency of 22 KHz, which received a mixture of DNA fragments ranging in size from 200 to >6000 BP (figure 1). The obtained DNA preparations were stored in the cold is through the chamber at ~18°C.
For formation of the complex with DNA solution was used Protamine sulphate 1% or 10 μg/μl for injection (herein, Moscow) and the DNA of the bacteriophage λ, gidralizovanny Hind III ("Medigen", Novosibirsk), or DNA of the human placenta, is fragmented to sizes from 200 to>6000 BP
In the study nuclease activity of the blood serum of mice after co-incubation of DNA and serum of mice were added proteinase K and incubated for 1 h at 65°C. After extraction with phenol/chloroform DNA was perioadele 0,6 V isopropanol in the presence of linear acrylamide and perform electrophoresis in agarose blocks.
In the experiments used cyclophosphamide (CP) production of JSC "Biochemist" (Saransk). The fit before applying was dissolved in saline and injected intraperitoneally at a dose of 200 µg/kg of body weight of the animal.
Blood cell count was taken from the tail individually each mouse immediately before the injection and at 4, 7 and 11 days after the administration of cyclophosphamide. Each experimental group consisted of 4 mice. The blood was diluted 20 times in 3% acetic acid solution and in the cell Goryaeva counted the number of cells in 1 mm3. The significance of differences between data was determined using t-test Student.
The study of the influence of DNA on the antitumor effect of cyclophosphamide was carried out on transplantable mouse is Oh lymphosarcoma LS and its subscheme RLS. LS was received by the employee ICG Vigledim induction nitrosomethylurea in mouse strain CBA, translated in ascitic form and supported on mice of this line. This tumor is highly sensitive to induction of apoptosis by cyclophosphamide and other alkylating agents. Tumor RLS obtained from LS by sequential intramuscular perepevok her relapses that occurred after exposure to increasing doses of cyclophosphamide, in consequence of which she acquired cross-resistance to induction of apoptosis and antitumor action of alkylating agents. Antitumor effect of different exposure was assessed by the inhibition of tumor growth and increased life expectancy carriers of tumour. The significance of differences between data was determined using t-test Student.
Cell culture adenocarcinoma human breast MCF-7 were cultured in medium RPMI 1640 with 10 mm L-glutamine and 50 μg/ml streptomycin Sigma (USA) at 37°C in the presence of 5% fetal bovine serum (FBS) "Bilat" (Russia) in an atmosphere with 5% CO2to a density of 0.7×107cells in 4 wells of a 24-hole tablet.
For preparation of DNA and its predecessor, 10 μg of DNA of the human placenta, is fragmented to sizes 200-6000 BP, were labeled nick-translation in the presence of a fragment of maple, three cold is one hot dNTP. To remove nevklyuchenie predecessors DNA was perioadele 2 times with isopropanol according to the procedure described by Glover [DNA Cloning. Methods. TRANS. from English. edited Dglover. M.: Mir, 1988. 538]. DNA was diluted in an appropriate volume of distilled water. DNA and Protamine were mixed in a 1:1 ratio by mass.
DNA samples were added to each well of the tablet (in the amount not exceeding 20 ál per well). In each well were 25-35 ml of medium. Of the sample, 1 µl of the working volume was selected on account of radioactivity.
In culture medium, in the hole where the cultivated cells were added to labeled DNA, processed as described above. Cells were incubated with DNA at 37°C, within the required time in an air thermostat. The incubation time was selected: 0' (labeled DNA was added in Wednesday, contained ice titrator, immediately after that, the medium was collected and added Triton X-100), 30'; 60'; 120'; 180'.
Upon completion of the incubation time the tablet was placed on ice. Wednesday was quantitatively collected and immediately added Triton X-100 in buffer A [Roberts DB. Drosophila: A Practical Approach. IRL Press, Oxford, 1986] (buffer contained 2 mm CaCl2, 0.5% Triton X-100). To each well was added 250 ál lyse buffer. The cells were left to lytic buffer on ice for 10'. Lysed cells resuspendable several times and was applied to a 1 ml gradient of 10% sucrose n is the buffer And, containing 2 mm CaCl2. Centrifugation was carried out in conical tubes (25 ml) in a bucket-rotor in the centrifuge C at 600g (2000 rpm) for 20 minutes. The supernatant representing zitoplazmaticescuu fraction, was collected in a separate tube and precipitated with isopropanol. Sediment cores were washed twice with 250 ál lyse buffer (buffer a containing 2 mm CaCl2, 0.5% Triton X-100). After each washing, the product was centrifuged 10' in the bucket-rotor in the centrifuge C at 600g (2000 rpm). Kernel resuspendable in 500 μl of buffer a containing 2 mm CaCl2analyzed cytologically and transferred into the tubes from the rotor JA-21 centrifuge J2-21. To a suspension of nuclei was added to 2 M NaCl and 1% SDS. The lysate cores were incubated at 65°15' to full enlightenment of the reaction mixture without any shaking. The lysate nuclei were centrifuged at 21000 rpm (52000 g) for 20 minutes at 35°C. the Supernatant, representing the nuclear juice or micromosaic material selected yellow nose dryness and transferred to another test tube. To the supernatant was added 1:10 volume of NaAc and besieged 0.6 volume of isopropanol. The amount of labeled material was determined in a standard way in appendrows tubes, placed in counting vials on the counter Rac Betta. Samples were fractionally in 0.7% agarose gel. After electrophoresis the agarose block was dried at padlock is under a stream of hot air. The gel was exposed to x-ray film during the night or depending on the amount of labeled material.
For analysis of DNA fragmentation during apoptosis cells MCF-7 were incubated 24 h at 37°C in the presence of 0.1 µg/ml of TNF-α and 10 µg/ml cycloheximide in medium RPMI 1640 containing 10 mm L-glutamine, 50 μg/ml streptomycin and 5% ELS. For DNA extraction to resuspending in water, the cells were added SDS to 1%, EDTA to 20 mM and PrK to 200 μg/ml and incubated at 60°C for 1 h DNA precipitated with isopropanol, dissolved in water and the newly added EDTA to 20 mM and PrK to 200 μg/ml and incubated at 60°C for 1 h, the Samples were analyzed in 2% agarose gel electrophoresis.
Consider leucotonalite action fragments of exogenous DNA that is protected by Protamine, when experimentally induced fit mielosupression mice.
Will focus on the dynamics of degradation of exogenous DNA associated with Protamine and without the effects of nucleases in the serum.
Any unprotected DNA, caught in the blood, almost instantly exposed to nucleases, which leads to its degradation to fragments of sizes that are multiples of 1-2 nucleosomal the monomers and the components of ~200 BP [Cherepanova AV, Tamkovich, S.N., Vlasov V.V., Laktionov P.P. Activity desoxyribonuclease blood in norm and at a pathology. Biomedical chemistry. 2007. T, No. 5, s-496]. For the achievement of the possible pleiotropic [Nikolin VP, Popova N.A., Sebelia IE, Strunkin D. N., Rogachev V.A., Semenov D.V., S. Bogachev, Yakubov L.A., Shutov M.A. the Effect of exogenous DNA on the growth of experimental tumors. Oncology issues. 2006. T, No. 1, p.66-69; Nikolin V.P., Popov N.A., Sebelia IE, Strunkin D. N., Rogachev V.A., Semenov D.V., S. Bogachev, Yakubov L.A., Shutov M.A. the Effect of exogenous DNA on the restoration of leucopoiesis and antitumor effect of cyclophosphamide. Oncology issues. 2006. T, No. 3, s-340; Likhacheva AS, Nikolin VP, Popova NA, Dubatolova TD, Strunkin DN, Rogachev VA, Sebeleva THOSE Erofeev IS, Bogachev SS, Yakubov LA, Shurdov MA. Integration of human DNA fragments into the cell genomes of certain tissues from adult mice treated with cytostatic cyclophosphamide in combination with human DNA. Gene Ther Mol Biol. 2007; 11: 185-202; Likhacheva AS, Nikolin VP, Popova NA, Rogachev VA, Prokhorovich MA, Sebeleva THOSE Bogachev SS, Shurdov MA. Exogenous DNA can be captured by stem cells and be involved in their rescue from death after lethal-dose γ-radiation. Gene Ther Mol Biol. 2007; 11: 305-314; Yakubov LA, Rogachev VA, Likhacheva AC, Bogachev SS, Sebeleva THOSE Shilov AG, Baiborodin SI, Petrova NA, Mechetina LV, Shurdov MA, Wickstrom E. Natural human gene correction by increasing interest among small genomic DNA fragments. The Cell Cycle. 2007 Jul; 6 (18): 2293-2301] therapeutic effect associated with the amount of input DNA that is involved then in various types of reparative homologous recombination of exogenous DNA introduced into the body, amounted to a total of from 0.3 to 6.25 mg per animal, which greatly exceeded the physiological norm.
Initially, we assessed nuclease activity of serum. As the substrate was used a gene is MNA human DNA, isolated from placentas of healthy parturients, fragmented to sizes from 200 to >6000 BP was studied the dynamics of degradation of human DNA in the serum of mouse blood at concentrations of 1, 10, 50 and 97%. The mixture was incubated 10 min at 37°C.
As can be seen from the obtained results, after 10 min incubation in 1% serum noticeable degradation of the original DNA. At all other concentrations of serum in the reaction mixture, the original DNA is degraded to fragments of size ~200 BP (figure 2).
To prevent degradation currently actively developed different methods of DNA delivery into the cell, for example the use of DNA-binding proteins in combination with ligands that selectively interacts with receptors on the surface of target cells, mediating endocytosis. To protect DNA from nucleases blood used viral vectors, liposomes and other [Anisimov, V.A. Targeted delivery of DNA vaccines. 8-th international conference "HIV Vaccine". 2000].
We hypothesized that the complex of DNA with Protamine will help to protect DNA from nucleases in the blood and thus will not reduce leucotonalite activity of the drug exogenous DNA, as described in our previous work [Nikolin V.P., Popov N.A., Sebelia IE, Strunkin D. N., Rogachev V.A., Semenov D.V., S. Bogachev, Yakubov L.A., Shutov M.A. VL is of exogenous DNA to restore leucopoiesis and antitumor effect of cyclophosphamide. Oncology issues. 2006. T, No. 3, s-340].
Protamine - low-molecular nuclear protein, molecular weight 4 to 12 kDa. For Protamine is characterized by a high content of alkaline amino acids, especially arginine (70-80%), which determines the basic properties of Protamine. It is well soluble in water, acidic and neutral medium, precipitated by alkalis, not denaturised when heated. Many animals Protamine along with histones is contained in the sperm, and some (such as fish) completely replaces the histones. In the nuclei of cells Protamine, like histones associated with deoxyribonucleic acid in nucleoprotein. The x-ray structure analysis shows that the chain of Protamine wound as the third thread around the DNA double helix. The presence of Protamine protect DNA from nucleases and gives the chromatin compact form. Protamine form salts with acids and complexes with acidic protein (soluble complex with Protamine insulin use in medical practice for renewal last) [L Brewer, Corzett M, Lau EY, Balhorn R. Dynamics of protamine 1 binding to single DNA molecules. J Biol Chem. 2003 Oct 24; 278 (43):42403-42408; Vilfan ID, Conwell CC, Hud NV. Formation of native-like mammalian sperm cell chromatin with folded bull protamine. J Biol Chem. 2004 May 7; 279 (19): 20088-20095].
To determine the ratio of DNA and Protamine, wherein the DNA molecule is fully protected from degradation, λ Hind III Dnce human DNA, associated with Protamine in different ratios, incubated in 10% serum of the mouse.
Initially, for the formation of the complex of Protamine/DNA 2 µg λ DNA Hind III and 15 μg of human DNA incubated for a few minutes with different (from 10 to 150% by mass) amount of Protamine. Further, the complexes Protamine/DNA obtained for different ratios of counterparties, incubated for 60 minutes in 10% serum of the mouse. As follows from the obtained data, Protamine in a ratio of 80% to the DNA mass almost completely protects DNA from degradation (figure 3).
Stop for stimulating recovery of leucopoiesis exogenous DNA associated with Protamine in a ratio of Protamine/DNA 0.8/1.0.
In this work, experiments were carried out in which for injection the mice used DNA associated with Protamine. We investigated the stimulation recovery of leucopoiesis after administration to mice of alkylating cytostatic of cyclophosphamide, inhibiting blood.
Cyclophosphamide (CP) - one of the most widely used drugs in Oncology practice. Antitumor effect of CPH currently associated with its genotoxic alkylating derivative of phosphoramide mustard (FM), which is spontaneously formed from aldophosphamide derived 4-hydroxy-fit. FM forms adducts with purine bases of DNA molecule, CA and neighboring guanine residues, and leads to crosslinking of double-chain DNA molecules [Yu LJ, Drewes P, Gustafsson K, Brain EG, Hecht JE, Waxman DJ. In vivo modulation of alternative pathways of P-450-catalyzed cyclophosphamide metabolism: impact on pharmacokinetics and antitumor activity. J Pharmacol Exp Ther. 1999 Mar; 288 (3): 928-937; De Silva JA, McHugh PJ, Clingen PH, Hartley JA. Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells. Mol Cell Biol. 2000 Nov; 20 (21): 7980-7990; Karle P, Renner M, Salmons B, Günzburg WH. Necrotic, rather than apoptotic, cell death caused by cytochrome P450-activated ifosfamide. Cancer Gene Ther. 2001 Mar; 8 (3): 220-230].
Cyclophosphamide induces education miaocheng links in actively dividing cells, resulting in die not only tumor cells, but also, for example, blood cells. As a result, at the organism level is observed radiation, the severity of which depends on the dose of cyclophosphamide.
Mice-male CBA/b was introduced cyclophosphamide 200 mg/kg of body weight. To count the number of cells for 30 min prior to this, all mice took the blood from the tip of the tail 10 µl. Similarly, the blood sampling was carried out on 4, 7 and 11 days after administration of CP. One group of mice served as a control (saline), and the other two next 3 days after administration of CP intraperitoneally injected 50 μg of human DNA - the same group, another human DNA in complex with Protamine.
As demonstrated by the results of the experiment (figure 4), on the 4th day, despite complete inhibition of the activity of the white sprout cu is VI, leukocyte count is stored in 2 times more than in the control group.
On day 7 after administration of CP, the number of leukocytes in mice, which drove human DNA or human DNA associated with Protamine, significantly higher than that in the control group, and even higher than the initial level.
On the 11th day, there is a slight natural decline in the number of cells, however, still recovering leucopoiesis in groups of mice that received additional injections of exogenous DNA, more effective than in the control.
This property exogenous DNA to stimulate leucopoiesis associated with multiple mechanisms of its effects on cell-precursor cells.
1. Exogenous DNA fragments are delivered in micromason space actively proliferating cells, including all predecessors of leucopoiesis [Histology, Cytology and embryology. Edited Afanasyeva SCI, Kuznetsova S.L., Yurina N.A. M.: Medicine, 2004. 768], and reparation miaocheng links (MDC)emerged under the influence of CP occurs with their participation. At the final stage of reparation MDC requires homologous recombination, for which donor sequences in normal conditions are homologous sites sister chromatids or homologous chromosomes. When multiple MDC process of reparation can be difficult steric about what Lema, arise when searching for homologous sequences and coupling circuits. Located in close proximity to repairwoman MDC exogenous fragments having homology with areas of repair, can be used as donor sequences and can participate in homologous recombination, required for completion of the repair process. In this part of exogenous DNA extrachromosomally localization served by a greater number repairwear damage that must surely affect the viability of the progenitor cells. The fact of the integration of exogenous DNA during repair MDC was described in [Likhacheva AS, Nikolin VP, Popova NA, Dubatolova TD, Strunkin DN, Rogachev VA, Sebeleva THOSE Erofeev IS, Bogachev SS, Yakubov LA, Shurdov MA. Integration of human DNA fragments into the cell genomes of certain tissues from adult mice treated with cytostatic cyclophosphamide in combination with human DNA. Gene Ther Mol Biol. 2007; 11: 185-202].
2. Another factor affecting the survival of Mature precursor cells leukocytes after exposure to ZF and processing of exogenous DNA, is described in the literature the phenomenon of the "opening of the genome", is characteristic for Mature progenitor cells, precursor cells or SK, which came in the way of terminal differentiation. This phenomenon is associated with the emergence of numerous functional single-stranded breaks chromatin that PR is palagay involvement of genetic material in reorganization, which is a mandatory and integral part of the differentiation and changes in the pattern of gene expression [Farzaneh F, Zalin R, Brill D, Shall S. DNA strand breaks and ADP-ribosyl transferase activation during cell differentiation. Nature. 1982 Nov 25; 300 (5890): 362-366; Johnstone AR, Williams GT. Role of DNA breaks and ADP-ribosyl transferase activity in eukaryotic differentiation demonstrated in human lymphocytes. Nature. 1982 Nov 25; 300 (5890): 368-370; Vatolin SY, Okhapkina EV, Matveeva NM, Shilov AG, Baiborodin SI, Philimonenko VV, Zhdanova NS, Serov OL. Scheduled perturbation in DNA during in vitro differentiation of mouse embryo-derived cells. Mol Reprod Dev. 1997 May; 47 (1): 1-10]. Splash lakirovanie DNA coincides with the first third-mitotic differentiated cells and defines a sharp increase (5-6 times) frequency of nursing chromatide exchanges. The proposed mechanism for this kind of exchange in the genome is homologous recombination [Vatolin SY, Okhapkina EV, Matveeva NM, Shilov AG, Baiborodin SI, Philimonenko VV, Zhdanova NS, Serov OL. Scheduled perturbation in DNA during in vitro differentiation of mouse embryo-derived cells. Mol Reprod Dev. 1997 May; 47 (1): 1-10]. Apparently, each stage of maturation of leukocytes characterized by the reorganization of chromatin and activation of a new ensemble of expressed genes. Presumably, the mechanism that determines the increased survival of progenitor cells after treatment fits and exogenous DNA, consists in the following. Cell-predecessor of leukocytes after last repair MDC in the presence of exogenous DNA extrachromosomally localization or without it retains a certain number of defects HRO is Atina, resulting from aberrations in the development and implementation of intermediates. That rests saved the cage with the remaining chromatin defects after repair MDC begins to divide. If the cell does not begin the reorganization of the chromatin associated with the transition of cells to the next stage of ripening, it inevitably dies due to defective mitosis. If the division is accompanied by a simultaneous transition of cells to the next stage of maturation, characterized by the reorganization of chromatin and the emergence of multiple single-stranded breaks, the presence in the nucleus fragments of exogenous DNA allows the cell to fix the remaining bugs and fully restore its viability. Apparently, there is some consistency between systems control the progression of the cell cycle and recombination-repair systems in the cell. It is assumed that the control checkpoint systems at the onset of functional breaks genome terminated or somehow changed. This allows fragments of exogenous DNA to remain unnoticed by the control systems and be involved in reparative-recombination processes associated with the restoration of the integrity of chromatin occurred after the reorganization of the genome as a homologous donor sequences.
3. The presence of aksogan the th DNA extrachromosomally localization in mikromazernom kernel space stimulates continuous proliferation of progenitor cells, precursor cells at all stages of maturation. The proposed mechanism of this stimulation is the following. As a result of hit in resting cell of an exogenous DNA fragments leads to the activation of checkpoint systems, controlling the progression of the cell cycle [MacDougall CA, Byun TS, Van S, Yee MC, Cimprich KA. The structural determinants of checkpoint activation. Genes Dev. 2007 Apr 15; 21 (8): 898-903; Yoo HY, Jeong SY, Dunphy WG. Site-specific phosphorylation of a checkpoint mediator protein controls its responses to different DNA structures. Genes Dev. 2006 Apr 1; 20 (7): 772-783; Yoo HY, Shevchenko A, Shevchenko A, Dunphy WG. Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses. J Biol Chem. 2004 Dec 17; 279 (51): 53353-53364; Zou L. Single - and double-stranded DNA: building a trigger of the ATR-mediated DNA damage response. Genes Dev. 2007 Apr 15; 21 (8): 879-885]. This enables certain programs, positioning the cell as arrested at an appropriate stage of the cell cycle. After activation of such programs cell is no longer able to block the cascade of events associated with this activation, and that rests before the cell starts to divide. If fragments of exogenous DNA each time enters into the internal compartments precursor cell, it will be without rest to share until such time as the stress factor is removed from its environment.
4. And the last one. Fragments of exogenous extracellular DNA act on all stages of maturation predecessors terminal differentiated blood cells in all areas of the bone marrow and thymus, 3 of which p is oshodi the formation of T-lymphocytes, and 4 is the maturation of b-lymphocytes. At every stage there is a certain number of cell divisions corresponding clone, leading to increased populations of these cells. After exposure to cytostatic cells of all stages of maturity takes time for completion of reparative processes and the continued maturation to the output terminal differentiated cells in the blood. In the case when the repair miaocheng crosslinking takes place without the participation of exogenous DNA, the majority of precursors at all stages of maturation dies. And recovery of the number of formed elements of blood begins with the survivors of the CCM. This requires time and effort of the organism to complete all stages of ripening and for all groups of the precursors of blood cells. The impact of exogenous DNA saves the population of cells at all stages of maturity. We believe that this fact explains the strong and fast-paced leucotonalite effect when exposed crosslinking cytostatic and therapy of exogenous DNA.
In work it is shown that after injection the mice alkylating cytostatic of cyclophosphamide and drug fragmented exogenous DNA associated with Protamine protein that protects DNA from nucleases in the blood, there is a effective recovery of lacopo the A.
The data obtained indicate that the use of DNA complexes with Protamine helps to protect DNA from nucleases blood without changing therapeutic activity of the drug with DNA.
DNA binding with Protamine can reduce the amount of input DNA and keep the original size of the fragments that encourages longer chromatin in reparative homologous recombination, ultimately, increasing the chance of the cells to get rid incurred multiple injuries.
Consider the action of exogenous DNA associated with Protamine, on experimental tumors of the mouse.
Will focus on the analysis of some quantitative and qualitative characteristics of exogenous DNA (unprotected Protamine and associated with him)delivered in experimental tumors.
To estimate the amount absorbed by the tumor DNA and morphology of DNA delivered in micromason space cells solid and ascitic forms of tumor RLS were conducted injections or DNA of the phage lebda, hydrolyzed Hind III, or human DNA fragmented to sizes 200-6000 P.O., mice with inoculated experimental tumors. As follows from the obtained data, the tumor is delivered to a small percentage of the injected DNA. When converted to BP about 1 fragm the t size of about 1.3 KBP may be present in the same cell tumors when injected up to 5 μg of exogenous DNA (figure 5, table 1-3).
The morphology of the labeled DNA extracted from blood plasma of mice, suggests that the blood line is almost instantaneous degradation of the original DNA (fig.6b, 12). In the blood plasma of mice carriers of tumour DNA is also subject to degradation. In degraded form of exogenous DNA is delivered into the internal compartments of a cancer cell (Figa).
After Association with Protamine injected DNA is protected from the action of nucleases blood and after 20 min in the blood, you can still find high-molecular fragments labeled DNA (fig.6b, 15).
|The amount of DNA that was injected into the mouse|
|the number of experiment||mcg||the expense of radioactivity (CPM)|
|Quantitative characteristics are delivered in some organs of the mouse labeled material (unprotected Protamine DNA) when administered intravenously|
|Experiment 1 (account frozen tissue)||Experiment 2||Experiment 3|
|time after injection of labeled DNA, min||0||30||60||120||180||180||180||180||180|
|2.47||0.16||0.11||0.14||0.12||0.0005||-||% added to the mouse|
|-||-||-||-||-||-||-||-||0.02||% added to the mouse|
|4.94||3.71td align="center"> 3.55||3.41||2.99||0.04||0.07||0.14||0.49||% added to the mouse|
|0.27||0.28||0.31||0.42||0.33||0.09||0.01||0.12||0.16||% added to YSL|
|The amount of DNA delivered into the cell tumors, in terms of HW / SW|
|the number of experiment||The size of the tumor, ál||The number of cells *109||DNA in the middle is delivered to the tumor, ng||DNA in the middle is delivered to the cell *10-9ng (*10-18g)||The proportion of the cellular DNA is delivered labeled DNA per cell *10-6||DNA is delivered to one cell, BP|
|4 (Protamine)||3000||3||10.80 (0 min)||3.6||0.6||3240|
|1 µl of the tumor contains 106cell|
|the mass of DNA in the cell the mouse 6 pg (6*10-12g)|
|haploid mouse genome is 2.7*109BP|
Stay on fragments of exogenous DNA associated with Protamine, on the growth of experimental tumors RLS.
As follows from the data obtained in the first part of the study, the tumor is delivered only small amounts of DNA. This allows to assume that the effect of inhibition of tumor growth shown in our earlier work [Yakubov LA, Popova NA, Nikolin VP, Semenov DV, Bogachev SS, Os'kina IN. Increasing interest among genomic DNA protects mice against radiation and chemical mutagens. Genome Biol. 2003; 5: 3; Nikolin V.P., Popov N.A., Sebelia IE, Strunkin D. N., Rogachev V.A., Semenov D.V., S. Bogachev, Yakubov L.A., Shutov M.A. the Effect of exogenous DNA on the growth of experimental tumors. Oncology issues. 2006. T, No. 1, p.66-69] and in the present study, not associated with direct exposure to fragments of exogenous DNA on the genome, as described for cancer MCF-7 cells growing in culture [Yakubov LA, Rogachev VA, Likhacheva AC, Bogachev SS, Sebeleva THOSE Shilov AG, Baiborodin SI, Petrova NA, Mechetina LV, Shurdov MA, Wickstrom E. Natural human gene correction by increasing interest among small genomic DNA fragments. The Cell Cycle. 2007 Jul; 6 (18): 2293-2301]. More likely the impact of fragments of exogenous DNA through certain parts of the immune system.
It is known that fragments of exogenous DNA or oligonucleotides enriched in GC motifs trigger DC maturation and induce an immune response in T-cell type. Apparently, even a very short DNA fragments having a specific structure, can affect antigenpresenting dendritic cells and activated T-cell immune response [Krieg AM. CpG motifs: the active ingredient in bacterial extracts? Nat Med. 2003 Jul; 9 (7): 831-835; Olishevsky S, Kozak V, Yanish Y. Immunostimulatory CpG DNA in cancer vaccinotherapy. Exp Oncol. 2003; 25: 85-92]. We believe that it is this path goes inhibition of tumor growth observed when injecting mice with inoculated tumors, fragm the appropriate exogenous DNA. In the case of a direct action on the genome using homologous recombination as a mechanism of substitution nkolokosa healthy allele, it is assumed that only long fragments (less than 1 KBP) can make this kind of recombination (double reciprocal exchange terminal homology) [Orr-Weaver TL, Szostak JW, Rothstein RJ. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci USA. 1981 Oct; 78 (10): 6354-6358; Kucherlapati RS, Eves EM, Song KY, Morse BS, Smithies O. Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA. Proc Natl Acad Sci USA. 1984 May; 81 (10): 3153-3157; Deng S, Capecchi MR. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol Cell Biol. 1992 Aug; 12 (8): 3365-3371; Thomas KR, Deng C, Capecchi MR. High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors. Mol Cell Biol. 1992 Jul; 12 (7): 2919-2923; Hastings PJ, McGill C, Shafer B, Strathern JN. Ends-in vs. ends-out recombination in yeast. Genetics. 1993 Dec; 135 (4): 973-980; Langston LD, Symington LS. Gene targeting in yeast is initiated by two independent strand invasions. Proc Natl Acad Sci USA. 2004 Oct 26; 101 (43): 15392-15397]. Apparently, in this case, there is a direct relationship, which is expressed in the fact that the longer the fragment, participating in GR, then the greater the likelihood of substitution nkolokosa not mutant allele and changes cancerous status of a cell. Assuming that both of these mechanisms are at the same time to inhibit the growth of cancer cells, the length of delivered fragments is an important factor for such inhibition.
In our work for t is rapie experimental cancers, we used a large number of momentary input DNA [Yakubov LA, Popova NA, Nikolin VP, Semenov DV, Bogachev SS, Os'kina IN. Increasing interest among genomic DNA protects mice against radiation and chemical mutagens. Genome Biol. 2003; 5: 3; Nikolin V.P., Popov N.A., Sebelia IE, Strunkin D. N., Rogachev V.A., Semenov D.V., S. Bogachev, Yakubov L.A., Shutov M.A. the Effect of exogenous DNA on the restoration of leucopoiesis and antitumor effect of cyclophosphamide. Oncology issues. 2006. T, No. 3, s-340]. This was due to its rapid degradation in the blood stream and, as a consequence, the impossibility of getting into the tumor of long DNA fragments, which, as expected, along with the induction of the immune response, can replace netransformovany homologous areas of the genome and modify the genetics of the cancer cells. Association of DNA fragments with Protamine protects fragments from nuclease degradation. This high-molecular fragments reach micromanage space of the nuclei of cancer cells and can be used by the cell as a substrate for homologous recombination.
However, we found that when the number of input DNA, equal to a total of 120 μg per mouse, the tumor cells will be delivered no more than a few fragments of size 1 KBP (see previous section). This number most likely is not enough to affect the genetics of the cancer cells. We believe that the effect on the tumor in this case occurred through activation defined the components of the immune system. Important was the fact that associated with Protamine and preserved in this regard, the size of exogenous DNA inhibits tumor growth to the same extent as unprotected Protamine-DNA (7).
That is the main mechanism of inhibition of tumor growth at the Association of therapeutic DNA-Protamine is saved. Thus there is a possibility of a second mechanism of action of tumor, when long DNA fragment protected by Protamine, getting into a cancer cell, enters into homologous exchange of genomic DNA, which under certain conditions can affect the genetics of the cancer cells.
Consider the internalization in MCF-7 cells exogenous DNA fragments protected by Protamine and the induction of their apoptosis.
Stay on comparative morphological characteristics of the fragments of exogenous DNA internalized in the main cell compartments of MCF-7 cells from the culture medium in unprotected form and associated with Protamine.
On Fig presents the results of electrophoresis of DNA extrachromosomally separated from the nuclei of MCF-7 cells.
As follows from the presented electrophoregram DNA samples, the Association added to the medium of exogenous DNA with Protamine, mikromazernom space nuclei of cells detected train labeled DNA stretching the I from the low molecular weight fractions up to high molecular weight, that may be evidence of delivery in the nuclear space of the entire spectrum (200-6000 BP) fragments of culture medium. High-molecular fragment part of the experiment with unprotected DNA detected in microsomes faction, is the result of ligation of short degraded fragments, delivered in the nuclear space [Rogachev VA, Likhacheva A, Vratskikh Oh, Mechetina LV, Sebeleva THOSE Bogachev SS, Yakubov LA, Shurdov MA. Qualitative and quantitative characteristics of the increasing interest among DNA delivered to the nucleus of a living cell. Cancer Cell Int. 2006 Oct 11; 6:23]. Also on electrophoregram DNA samples isolated from the culture medium after various times of incubation, when the Association with Protamine in an environment that has high molecular weight form of DNA (data not shown). However, the samples with DNA added to the medium in the form of, unassociated with Protamine, she cavity degrades [Rogachev VA, Likhacheva A, Vratskikh Oh, Mechetina LV, Sebeleva THOSE Bogachev SS, Yakubov LA, Shurdov MA. Qualitative and quantitative characteristics of the increasing interest among DNA delivered to the nucleus of a living cell. Cancer Cell Int. 2006 Oct 11; 6:23].
The dynamics of the delivery of fragments of exogenous DNA into the internal compartments of the cell are presented in table 4 and figure 9.
|The amount of labeled DNA, detective in culture medium and in vnutrineironalnah cells, after incubation, the culture of MCF-7 cells with intact and connected with Protamine DNA during various time|
|DNA||0||30'||60'||120'||180'||the incubation time, min|
|0.2501||0.7954||1.4808||1.8433||1.3573||% of added|
|0.3461||0.9557||1.2425||1.0367||1.1414||% of added|
|0.0065||0.0255||0.0097||0.0073||0.0348||% of added|
|0.2654||0.4490||0.5440||0.4981||0.1577||% of added|
|DNA + Protamine||0||30||60||120||180||the incubation time, min|
|0.6528||2.1021||5.8305||6.7990||4.7599||% of added|
|0.4083||1.7083||1.1009||1.6427||1.2018||% of added|
|0.0127||0.1383||0.2107||0.0767||0.3244||% of added what about the|
|0.0048||0.1131||0.2226||0.4830||0.1252||% of added|
We can note some features of the internalization of exogenous fragmented DNA that is protected by Protamine.
1. There are two peak internalization of fragments of exogenous DNA in the cytoplasm at the points 30' and 120'.
2. In micromason space faction, these peaks are shifted to points 60' and 180'.
3. In micromason space is delivered almost 10 times the quantity of exogenous DNA that is protected by Protamine, compared to the unprotected DNA.
4. Dynamics of appearance of labeled material in the fraction of chromatin differs significantly in the case of DNA associated with Protamine, and without it.
In the faction of chromate what and when using unprotected exogenous DNA labeled material is found in a significant number of already at the zero point. At points 30-120' the number of labeled material slightly increases and reaches a plateau. At the point 180' number of labeled material in the chromatin fraction drops sharply. In the case of protected Protamine DNA process has an explicit cumulative. With virtually no zero point labeled material accumulates at a point 120' reaches its maximum value, matching a DNA sample without Protamine. Then the number of labeled material will be reduced to the same values as in the DNA sample used without Association with Protamine.
The detected difference in the amount of delivered material DNA in micromemo fraction can be explained by the fact that the receptors that bind DNA at the cell surface, have greater specificity to DNA in the form of chromatin, than an unprotected DNA. Two peaks in the number of labeled material detected in the cytoplasm of 30' and 120' and microsomes faction 60' and 180'may indicate that the number of receptors the end that they, together with DNA internalisers in cellular compartments that the turnover of the receptors occurs within approximately one hour and that the penetration of labeled fragments from the cytoplasm into the inner space kernel takes about 30 minutes. Differences in the dynamics of appearance of labeled material in the fraction of chromatin can be made the thread topics when using unprotected DNA released into the nuclear space labeled fragments rapidly undergo hydrolysis and labeled precursors are involved in the replication process. In the case of DNA protected by Protamine form, although in mikromazernom space accumulates a large number of fragments of exogenous DNA, continued throughout the analyzed time 0-180', chromatin is detected by sequentially increasing the number of labeled material. It can be assumed that such dynamics can be involved in two processes that can act separately or superimposed on one another. One of them, similar to those described for unprotected DNA and is associated with the degradation of labeled fragments and the use of labeled precursors in the synthetic processes in the cell. You can also assume that the fragments of exogenous DNA that is protected by Protamine and deposited in mikromazernom space, gradually recombine with homologous areas chromium, which leads to a gradual accumulation of label in the chromatin.
Will focus on the analysis of apoptosis induction fragments of exogenous DNA that is not protected by Protamine and in complex with him.
We conducted experiments on induction of apoptosis of MCF-7 cells treated with DNA, the Conn is Noah with Protamine at a concentration of 25 μg/ml and unprotected DNA at a concentration of 100 µg/ml As follows from figure 10, the DNA is protected by Protamine, equally induces apoptosis, as unprotected DNA.
This fact may indicate that the protected DNA is also able to engage in reparative homologous recombination, correcting the mutant allele, while its effective amount in the culture medium can be significantly lower.
In addition to the analysis of apoptosis induction by DNA fragments protected by Protamine, in comparison with the unprotected DNA, we conducted experiments in which the concentration of the protected DNA in the culture medium was reduced to such designated for the blood plasma of higher eukaryotes 30-100 ng/ml [Anker P, Mulcahy H, Chen XQ, Stroun M. Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients. Cancer Metastasis Rev. 1999; 18 (1):65-73].
In cell culture MCF-7 was added to the unprotected DNA and DNA associated with Protamine, at concentrations of 25, 2.5, 0.25 and 0.025 ág/ml of medium. The number of cells for all experimental points were the same (107). After 17 days of continuous incubation of cells with DNA cell culture induced apoptosis, were isolated DNA and watched the emergence of nucleosomal fragmentation of chromatin (11).
It was found that the concentration of DNA associated with Protamine, 25 ng/ml of medium is able to cause nucleosomal fragmentation of chromatin.
Protamine, protecting DNA from the action of h is of cleas, contributes to the fact that more DNA enters the cell in non-degraded form, and accordingly is able to participate in the recombination process. This suggests that a greater number of cells restores gene caspase 3, and induction of apoptosis, we can observe a more pronounced nucleosomal fragmentation of chromatin.
Thus, the binding of Protamine provides DNA molecules resistance to nucleases found in the culture medium. As a result, fragments of exogenous DNA can reach the main cellular compartments in its original form. Under cultivation of MCF-7 cells together with DNA associated with Protamine, recovery has been observed gene caspase 3, which is phenotypically manifested in the emergence of nucleosomal degradation of chromatin in the induction of apoptosis. Protamine, protecting the DNA from nucleases, provides the ingress of exogenous DNA molecules per cell in non-degraded form, which makes possible their participation in the recombination process. When linking DNA-Protamine becomes possible to reduce the concentration of DNA without affecting the efficiency of exogenous DNA.
Thus, thanks to usovershenstvovaniju known method achieved the desired result, which consists in increasing efficiency is ecene patients passing software chemotherapy, and patients with cancer. The results of this study allow us to say that the protection of fragmented exogenous DNA gestonorone protein Protamine does not affect the ability fragmented exogenous DNA to be leucotonalite action to inhibit the growth of experimental tumors of the mouse and to participate in the reparations mutant allele of caspase 3 cells adenocarcinoma human breast MCF-7. Association fragmented exogenous DNA with Protamine allows you to keep the original size of therapeutic fragments and significantly reduce the effective concentration of exogenous DNA into the extracellular fluids.
1. A method of treating cancer, comprising introducing into the patient a fragmented human DNA, characterized in that the fragmented human DNA is administered in combination with nonhistone protein Protamine, and the length of the DNA fragments is 200-6000 base pairs and the ratio of fragmented human DNA/nonhistone protein Protamine is 1/0,8, the complex is administered in a dose to the concentration of fragmented human DNA in the blood was 25 - 1000 ng/ml.
2. The method according to claim 1, characterized in that the introduction of the manufacture of parenteral or microcl what smerovanie.
SUBSTANCE: invention relates to a compound of formula Ia: and its pharmaceutically acceptable salt, where: p equals 0 or 1; n assumes values from 1 to 3, q equals 1; R5 is selected from hydrogen, -XNR7R8, pyrimidine-C0-4alkyl, pyridine-C0-4alkyl, phenyl, C3-10cycloalkyl-C0-4alkyl and C3-6heterocycloalkyl-C0-4alkyl, where C3-6heterocycloalkyl is a saturated monocyclic ring system containing the said number of atoms, provided that one or more of the said carbon atoms is substituted with O or NR, where R is hydrogen or C1-4alkyl; R7 and R8 represent C1-4alkyl; R6 denotes hydrogen; or R5 and R6 together with a nitrogen atom to which they are both bonded form morpholine or piperidine; where any piperdine-C0-4alkyl, piperidine-C0-4alkyl or C3-10cycloalkyl-C0-4alkyl of substitute R5 or a combination of radicals R5 and R6 can be optionally substituted with 1-2 radicals which are independently selected from -XNR7R8 and -XOR7, the said phenyl of substitute R5 is substituted with a -XR9 group, the said C3-6heterocycloalkyl-C0-4alkyl of substitute R5 is optionally substituted with a -XOR7 group, where X is a single bond or C1-4alkylene; R7 and R8 are independently selected from hydrogen and C1-4alkyl; R9 is selected from C3-10heterocycloalkyl which is a saturated monocyclic ring system containing the said number of atoms, provided that one or more of the said carbon atoms is substituted with O or NR, where R is as given above; R10 denotes hydrogen; R15 is selected from halogen, C1-6alkyl and C1-6alkoxy; and R16 is selected from halogen, methoxy, nitro, -NR12C(O)R13, -C(O)NR12R12, -NR12R12, -C(O)OR12 and -C(O)NR12R13; each R12 is selected from hydrogen and C1-6alkyl; R13 is selected from phenyl, thienyl, pyrazolyl, pyridinyl or isoxazolyl, where any phenyl, thienyl, pyrazolyl, pyridinyl or isoxazolyl of substitute R13 can be optionally substituted with 1-2 radicals which are independently selected from halogen, C1-6alkyl, halogen-substituted C1-6alkyl, imidazole-C0-4alkyl, C3-10cycloalkyl, C3-10heterocycloalkyl-C0-4alkoxy and C3-10heterocycloalkyl-C0-4alkyl; where the said C3-10heterocycloalkyl-C0-4alkoxy and C3-10heterocycloalkyl-C0-4alkyl each represent a saturated monocyclic ring system containing the said number of atoms, provided that one or more of the said carbon atoms is substituted with O or NR, where R assumes values given above; and the said C3-10heterocycloalkyl-C0-4alkoxy and C3-10heterocycloalkyl-C0-4alkyl can each be optionally substituted with 1 radical independently selected from C1-6alkyl, hydroxyl-substituted C1-6alkyl and NR7R8, where R7 and R8 assume values given above. The invention also relates to pharmaceutical compositions containing the said compounds.
EFFECT: obtaining novel compounds and compositions based on the said compounds which can be used in medicine for treating and preventing diseases or disorders associated with abnormal or uncontrolled kinase activity, particularly diseases or disorders associated with abnormal activity of kinase c-Src, FGFR3, KDR and/or Lck.
12 cl, 1 tbl, 2 ex
SUBSTANCE: invention refers to medicine and biotechnology and concerns an anticancer drug based on nanoparticles bearing recombinant human tumour necrosis factor alpha. Substance of the invention includes the anticancer drug representing nanoparticles each of which contains a nucleus consisting of polynucleotide complex representing double-helical RNA (dhRNA) - an interferonogenesis inducer, and coated with a layer of spermidine conjugate with polyglucin held by ionic interaction between negative polynucleotide complex and positive spermidine, while recombinant human tumour necrosis factor alpha is covalently bound with activated polyglucin. As double-helical RNA, the anticancer drug contains double-helical RNA of Saccharomyces cerevisiae yeast. Nanoparticles are ball shaped and sized about 50-70 nm; 60-80 molecules of recombinant human TNF-α of cytolytic activity 106 ME/mg of protein and higher, 60-80 molecules of polyglucin and 1000-1300 molecules of spermidine are necessary for one molecule of double-helical RNA of Saccharomyces cerevisiae yeast.
EFFECT: reduced dose of TNF-α and lower toxicity.
5 cl, 5 ex, 4 dwg
SUBSTANCE: invention refers to preparation of a plant drug for treating stomach cancer. The drug for treating stomach cancer contains cod-liver oil, badger fat, plant seed blood chosen from the group: pomegranate, hot pepper, fennel and activated coal in the following ratio, wt %: cod-liver oil 28; badger fat 28; seed blood 42; activated coal the rest.
EFFECT: treatment with the declared drug involves activating body defences, resistance ensured by improved functioning of an individual's organs and systems, and targeting the lesion focus.
SUBSTANCE: invention concerns medicine, namely oncology and can be used in mesothelioma treatment. The method consists in monthly subcutaneous introduction to the patient of powdered fibrin solution recovered from the donor's blood clot in a dose 100-600 mg of dry powder per one introduction session.
EFFECT: application of the invention allows for effective cancer therapy by fibrin preparation only without administration of cytostatic agents, radioactive drugs and radioisotopes that ensures reduced complications.
3 dwg, 1 ex
SUBSTANCE: invention describes a biomarker intended for determining sensitivity of proliferative diseases, such as cancer, to mTORs inhibitor combined with a cytotoxic agent, first of all with a cytotoxic agent (CA) that damages or disturb DNA integrity. According to the invention, the biomarker designated as p21 represents cip/kip-family of cyclinkinase inhibitors. Sensitivity or response of the proliferative disease in an individual on treatment with mTOR inhibitor combined with CA is determined by the level of p21 expression after CA treatment and a combination therapy with using CA and mTOR inhibitor. Favorable treatment and sensitivity of disease to the combination therapy is predicted by the absence of expression induction reduced. Besides according to the invention, the biomarker can be used in the method to overcome the CA resistance in the patient treated with CA. That is ensured by evaluating p21 level in a sample, the increasing regulation of p21 expression following CA introduction to the patient, mTOR inhibitor is administered in combination with CA, while lowered expression regulation observed following the combination therapy ensures to continue treatment with mTOR inhibitor with simultaneous or consecutive CA introduction.
EFFECT: application of the invention allows for more accurate prediction of sensitivity of a proliferative diseases in an individual to the combination therapeutic treatment.
4 cl, 5 ex
SUBSTANCE: invention relates to novel substituted quinoline derivatives of general formula (I) in which: m is an integer from 0 to 3; R1 is selected from a group comprising an acylamino group, ester carboxylic group and an alkyl with 1-5 carbon atoms, an optionally substituted hydroxy and a halogen; R2 denotes hydrogen or an alkyl with 1-5 carbon atoms; R3 denotes - C(=X)-A, where A is selected from a group comprising aryl, heteroaryl, heterocyclyl and cycloalkyl, each of which can optionally contain from 1 to 4 substitutes selected from a group comprising an alkyl with 1-4 carbon atoms, an alkoxy with 1-4 carbon atoms, a halogen, hydroxy or nitro, and X denotes oxygen or sulphur; R4 denotes alkylene-heterocyclyl or alkylene-NR7R8, where alkylene is a linear alkylene with 1-4 carbon atoms; R7 and R8 are independently selected from a group comprising hydrogen, an alkyl with 1-4 carbon atoms, arylalkyl, heteroarylalkyl, cycloalkyl or cyclo-alkylalkyl; R5 is selected from a group comprising L-A1, where A1 is selected from a group comprising aryl, heteroaryl, heterocyclyl and cycloalkyl, each of which can optionally contain from 1 to 4 substitutes selected from a group comprising an alkyl with 1-4 carbon atoms, an alkoxy with 1-4 carbon atoms, a halogen, hydroxy and nitro, and where L is selected from a group consisting of oxygen, -NR9, where R9 denotes hydrogen or alkyl; -S(O)q-, where q equals 0, 1 or 2, and an alkylene with 1-5 carbon atoms, optionally substituted with a hydroxy, halogen or acylamino; and R6 is selected from a group comprising an alkyl with 1-5 carbon atoms, an alkenyl with 2-5 carbon atoms, an alkynyl with 2-5 carbon atoms, -CF3, an alkoxy with 1-5 carbon atoms, a halogen and a hydroxy; or its pharmaceutically acceptable salts or esters, as well as to a pharmaceutical composition having anticancer activity or inhibitory effect on mitotic kinesin based on the said compounds, to a method of treating disorders and use of these compounds for making a medicinal agent.
EFFECT: novel compounds which can be useful in treating cancer are obtained and described.
40 cl, 3 ex, 1 tbl
SUBSTANCE: invention describes novel derivatives of 2,6-diaminopyridine of formula (I), where R1 is piperidine which is optionally substituted with up to four substitutes independently selected from a group comprising: (a) hydrogen, (b) lower alkyl, (c) lower alkyl substituted with an oxo group or aryl, (d) CO2R7, (e) COR12, (f) C(O)NR13R14, and (g) S(O)nR15; R2 is phenyl which can be substituted with up to four substitutes independently selected from a group comprising: (a) lower alkyl, (b) lower alkyl substituted with a halide or OR10, (c) halide, or (d) OR12; R5 and R6 are independently selected from a group comprising (a) hydrogen and (b) lower alkyl; R7 is selected from a group comprising (a) hydrogen and (b) lower alkyl; R10 is selected from a group comprising (a) lower alkyl, (b) aryl and (c) aryl substituted with a halide or NR5R6; R12 is selected from a group comprising (a) hydrogen and (b) lower alkyl; R13 and R14 are independently selected from a group comprising (a) hydrogen and (b) lower alkyl, R15 is selected from a group comprising (a) aryl, (b) aryl substituted with a halide, CO2R12, SO2R10, COR12, lower alkyl or lower alkyl substituted with a halide, OR12, oxo group, CO2R12, C(O)NR5R6 or NR5R6, (c) heteroaryl, (d) heteroaryl substituted with a halide, CO2R12, SO2R10, COR12, lower alkyl and lower alkyl substituted with a halide, OR12, oxo group, CO2R12, C(O)NR5R6 or NR5R6, (e) NR5R6, (f) lower alkyl, (g) lower alkyl substituted with a halogen, OR12, oxo group, CO2R12, C(O)NR5R6 or NR5R6, (h) a heterocycle and (i) a heterocycle substituted with CO2R12, COR12, SO2R10, lower alkyl, C(O)NR5R6 or NR5R6; n equals 0, 1 or 2; as well as a pharmaceutical composition having inhibitory effect on cyclin-dependant kinase and a method of producing the compound of formula I.
EFFECT: novel compounds which have antiproliferative activity and can be used for treating or curbing cancer are obtained and described.
28 cl, 58 ex, 4 tbl
SUBSTANCE: invention refers to 3-oxo-28-(N-methylpiperazine)-carbonyl-lup-20(29)-ene of formula (I) which can be used in medicine as a corrective agent for paraneoplastic damages and toxic effects of cytostatic polychemotherapy. .
EFFECT: compound I shows an apparent anticancer activity, reduces severity of pathological changes in tissues caused by paraneoplastic syndromes; in cytostatic polychemotherapy, it exhibits an apparent antioxidant and cytoprotective effect in normal cells of viscera and thereby does not stimulate proliferation and dissemination of a tumour.
8 ex, 8 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: there is described an immunogen for making an immunogenic cancer composition free of DNA-binding function and all domains of a zinc finger, on the basis of polynucleotide coding a nonfunctional mutant form of a related molecule ("brother") of regulator of imprint sites (BORIS) of protein, polypeptide or peptide, containing amino acid sequence presented in the description. The immunogenic cancer composition contains aforementioned immunogen and an adjuvant chosen particularly from cytokine, chemokin, a costimulating molecule. There is described an expression vector containing polynucleotide, coding above-stated protein, e.g., in bacterial systems, mammal systems, in yeast or viral systems. The cancer vaccine under the invention contains polynucleotide (immunogen), additionally the adjuvant and, if necessary, a pharmaceutically acceptable carrier. The invention describes the method for of cancer immunisation of a mammal with using said immunogen on the basis of polynucleotide.
EFFECT: invention allows improving effectiveness of cancer prevention.
28 cl, 7 dwg, 2 tbl, 1 ex
SUBSTANCE: invention covers medical products which contain pharmacologically acceptable liquid crystals and their derivatives of general structural formula: , where X represents a group -CH=N-, while Y and Z are alkyl or alkoxyl group and are taken in a daily dose in amlount 355 mcg to 10 g a day.
EFFECT: medical products under the invention exhibit antiinflammatory, immunomodulatory, analgetic (anaesthetising) and antineoplastic action when administered in optimal doses without foreign substances.
11 cl, 18 ex
SUBSTANCE: there is offered application of group of survival-improving polypeptide cone cells originated from rod cells and designated as RDCF, and also coding molecules of nucleic acid to prepare medicines, particularly pharmaceutical compositions used to treat retinal dystrophy. Methods for preparing RDCF by recombinant DNA technologies, and required aids, as well as preparation of antibodies distinguishing said polypeptides are described.
EFFECT: improved clinical effectivenesses.
12 cl, 19 dwg, 1 ex
SUBSTANCE: invention represents plasmid vector for transfer of DNA, which comprises sequence coding various fragments of oncoprotein p185neu, which are able to induce immune response in respect to tumors, which hyperexpress p185neu. Invention is also related to pharmaceutical composition on the basis of vector for prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.
EFFECT: invention makes it possible to increase efficiency of prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.
10 cl, 14 dwg, 2 tbl, 2 ex
SUBSTANCE: there is offered molecule of nucleic acid inducing CEA immune response, containing a nucleotide sequence that codes a fused protein on a basis of carcinoembryonal antigen (CEA) or its functional version fused with a subunit B of thermolabile enterotoxin E coli. There are described versions thereof, as well as the related purified protein. There is disclosed an expression vector containing said molecule of nucleic acid, and a host-cell containing specified vector. There are described adenoviral vaccinal vector for inducing the immune response and a vaccinal plasmid on the basis of the specified molecule.
EFFECT: application of the invention allows to inducing the immune response in a mammal which is stronger, than that induced with natural CEA that can find application in medicine for cancer treatment.
20 cl, 62 dwg, 20 ex
SUBSTANCE: cardiac hystiocytes are proliferated by inducing of cyclin and CDK expression in cardiac hystiocytes and by inhibiting function or activity of protein of Cip/Kip family or by inhibiting production of protein of Cip/Kip family. There is described expression vector that contains a cyclin gene, a cyclin-dependent kinase gene and one or several agents chosen from the group consisting of a gene coding the factor which inhibits production, function or activity of protein of Cip/Kip family and nucleic acid sequence which inhibits production of protein of Cip/Kip family. There is disclosed pharmaceutical composition containing said vector, and applied for treatment of cardiac diseases. There is offered cardiac hystiocyte produced by the declared method. There is presented method of treating a cardiac disease that implies injection of the disclosed pharmaceutical composition.
EFFECT: regeneration and recovery of cardiac hystiocytes.
31 cl, 13 dwg, 1 tbl, 6 ex
SUBSTANCE: invention is related to biotechnology, namely to method for production of chondro-osteogenous cells in vitro and their application. Chondro-osteogenous cells are produced from mesenchyme stem cells of a human being. Mesenchyme stem cells of a human being are cultivated in medium with additives of human blood serum and beta -1 transforming factor of growth, having molecular domain, which provides for interaction with collagen I (TGF-β1-CBD). Then stem cells are exposed to further proliferation by means of addition of human blood serum and TGF-β1-CBD. In the end chondro-osteogenous induction is carried out by dexamethasone and -β-glycerophosphate. Chondro-osteogenous cells may be used in composition that is able to induce osteogenesis.
EFFECT: invention makes it possible to make medical preparations for recovery of bone and/or chondral tissue.
20 cl, 3 dwg, 3 tbl, 7 ex
SUBSTANCE: invention refers to medicine, namely to genetic therapy and concerns nucleotide sequence that codes insulin-like human growth factor IGF-1 presented with a synthetic gene including a sequence SEQ ID NO:1, recombinant plasmid DNA, containing this sequence, an eukaryotic cell containing recombinant plasmid DNA, construction for genetic therapy and a pharmaceutical composition for genetic therapy with regenerative and wound healing action.
EFFECT: advantage of the invention consists in decreased doses and introduction rate of injected preparations.
5 cl, 2 ex, 2 tbl, 4 dwg
FIELD: chemistry, biochemistry.
SUBSTANCE: invention refers to biotechnology, specifically, to cell differentiation and can be used in medicine. Differentiated stromal cell is made of adipose tissue. This cell expresses one or more proteins, characteristic for endocrine pancreas cell, such as insulin, glucagon, somatostatin and pancreatic polypeptide.
EFFECT: production of differentiated and functionally relevant cell for researches, implantation, transplantation and development of tissue products for treating pancreas diseases and repairing pancreas tissues.
24 cl, 8 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, in particular to hepatic cells production, and may be used in medical science. From the whole liver or resected part thereof, a cell population enriched with living cells of human liver, including hepatic stem cells/precursor cells, is obtained. Cell population contains functional hepatocytes and biliary cells expressing cytokeratin 19 (CK19), but not expressing albumin, as well as hepatic stem cells/precursor cells 9 to 13 mcm in diameter and expressing EP-CAM, CD 133 markers. Resulting cell population is used for hepatotherapy.
EFFECT: production of living population of hepatic cells sufficiently efficient for regeneration.
60 cl, 16 dwg
FIELD: medicine; oncology.
SUBSTANCE: method is based on introduction in patient's organism of cytostatic preparation with mechanism of action based on interchain cross-linking within DNA molecule of cancer cells with preparation of fragmented allogen DNA with the fragments of biologically active dimension and composing entire gene of physiologically and genetically healthy donor.
EFFECT: method enables to activate recombination cell system and to provide homologous integration of DNA preparation into recipient gene.
3 cl, 5 dwg, 1 tbl, 1 ex
SUBSTANCE: substance of invention includes separated population of hematopoetic stem cells of negative lineage, (Lin- HSC), containing progenitor endotheliocytes (EPC), protecting retina blood vessels and neuronal eye networks, method of separation of hematopoetic stem cell population of negative lineage from bone marrow of mammal adult, herewith approximately 20% of cells in separated Lin- HSC express cell surface CD31.
EFFECT: possibility to apply for treatment of eye vascular diseases.
22 cl, 10 ex, 24 dwg
FIELD: biotechnology, veterinary science.
SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.
EFFECT: valuable biological properties of recombinant virus.
41 cl, 13 dwg