Growth inhibition of tumor cells by ectodomain of syndecan-1
(57) Abstract:The invention relates to medicine, in particular to Oncology, and for reducing the growth of tumor cells. To do this create in the extracellular environment of the specified cell concentration sindelarova of ectodomain 0.7-1.0 nm. For the treatment of tumors also offer pharmaceutical composition comprising as an active ingredient indiannavy ectodomain. The method allows to increase the effectiveness of inhibition of growth of malignant cells. 3 S. and 19 C. p. F.-ly, 13 ill. The present invention relates to the field of biology and therapy of cancer. Primarily, the present invention relates to a method of slowing or controlling the growth rate of cells, particularly cancer cells, as a result of exposure to such a cell an effective quantities of ectodomains part of syndecan-1. The method of the present invention facilitates and ensures the normalization of the growth rate and the state of differentiation of malignant cells.Description of the prior art
Cell differentiation is based on the selective use of genetic information, programmed by extracellular stimuli, which, for example, can unlucky. It is becoming increasingly evident that cell surface proteoglycans play an important role in the regulation of cell behaviour. Syndecan represent surface proteoglycans cells, which has been shown to be involved in the recognition matrix and the binding of the growth factor on the basis of which it is assumed that they are involved in the regulation of cells. Known sequences of human, mouse, rat and chumacero of syndecans. Recently, there have been reviews on syndecans (Jalkanen with TCS. in kN. "Receptors for extracellular matrix, ed. J. MacDonald and R. Mechem, Academic Press, San Diego, PP 1-37 (1991) and Bernfield O., al., Annu. Rev. Cell Biol. 8:365-393 (1992)).Syndecan-1 represents the most well-characterized cell surface proteoglycan (Saunders with al., J. Cell Biol. 108:1547-1556 (1989); Mali from al., J. Biol. Chem. 265:6884-6889 (1990)).In the international patent application WO 90/12033 disclosed amino acid sequence and the sequence of the corresponding cDNA molecules mouse syndecan-1. Diagnostic method for detection of transformed cells by detection of changes is an expression of syndecan in transformed cells, and this method is described in international kachestvennyh cells by inducing expression of syndecan in these malignant cells are described in the international patent application (PCT/FI93/00514).Brief description of drawings
Fig. 1 depicts the sequence of the human syndecan-1.Mugs: possible sites GAG connection; underlined by the long line: transmembrane domain; emphasized short line: signal aataa polyadenylation.In Fig. 2 shows the sequence of the mouse syndecan-1.Fig. 3 depicts schematized structure of the capsid protein of wild-type, bushmaster and ecto transfection structures. Construction of wild-type contains ectodomain mouse syndecan full length (Mali, M. al., J. Biol. Chem. 268:24215 (1993)). Beshvostye design is formed using siteprovides mutagenesis using the oligonucleotide with the formation of deletion mutant with a single arginine residue in the cytoplasmic domain, as described in the examples (Miettinen, N. M. with al., J. Cell Sci., in press (1994)). Ecoconstruction can also be obtained with the use described in the examples siteprovides mutagenesis using the oligonucleotide, and it has a stop codon in the protease-sensitive site, adjacent to the surface of the cells, the vertical lines indicate the projected sites GAG join and use the site, Chu is nanofluorescence localization of syndecan-1 on the cell surface.Fig. 5 - the number of secreted ectodomain of syndecan-1 air-conditioned environment of the clones Ecto cells (Ecto 15, 34, 2, and 23). Cells were cultured for two days in the presence of 10 nm testosterone and used ectodomain of syndecan-1 accumulated in the environment. The culture medium used directly. Samples were normalized to the number of cells and an equivalent number of slots was bottiroli on the membrane Hybond-H+. Ectodomain of syndecan-1 was determined as described in the examples, a method of enhanced chemiluminescence using 281-2 (Miettinen, N. M al. , J. Cell Sci., in press (1994)). Quantification was performed using an analytical system with a computer image (Imaging Research Inc.). Presents mean values and SEM for two parallel samples.In Fig. 6 depicts the organization of actin filament clones Ecto cells. Ecto cells were cultured in the presence of 10 nm testosterone and actin filaments were visualized using madaminkhodjaevna of phalloidin.In Fig. 7 depicts the formation of colonies anjaleoni clones in soft agar. Cells were cultured for 12 days 0.33% soft agar, DMEM+5% FCS in the presence of 10 nm testosterone, in sootvetstviiami of syndecan-1 (examples) from the conditioned medium of cells Ecto 2 on the growth of NMuMG and treated with testosterone (10Nm) cells S115 (S115+). 1500 cells were transferred to 96-well microtiter plates and the cells were cultured in the presence of DEAE-selected ectodomain of syndecan-1 to achieve 75-85% merge control (without ectodomain of syndecan-1) cells (four days for NMuMG cells and three days for cells S115+). Then the cells were fixed with 2% paraformaldehyde, stained with 0.5% of the dye crystal violet and washed with distilled water. Stained cells suspended in 10% acetic acid and the quantity was determined spectrophotometrically at a wavelength of 595 nm.In Fig. 9 shows the effect heparinases processing DEAE-selected ectodomain of syndecan-1 on inhibition of cell growth S115+. Cells S115+ were cultured in the presence of 1 nm DEAE-dedicated syndecan-1 of culture medium Ecto 2 cells and from the environment NMuMG cells or in the presence of such preparations pretreated with heparinase (Seikagaku Kogyo To.) for 1 hour at 37oC.In Fig.10 shows the effect of purified immuno-adsorption method ectodomain of syndecan-1 on cell growth S115+ and NMuMG. DEAE-selected ectodomain of syndecan-1 was further purified on 281-2 immunoaffinity column (examples). Cells S115+ and NMuMG were cultured in the th ectodomain of syndecan-1, not HS or CS GAG inhibits the growth of cells S115+.In Fig. 12 shows the inhibition of growth of cells of different cell lines (CarB, MCF-7, S115+ in the presence of 10 nm testosterone, S115+ without testosterone, NIH T, NMuMG and Nasal) using 1 nm DEAE-selected ectodomain of syndecan-1 (examples). Cell growth was analyzed on all panels similarly shown for panels (a) and were compared with the behavior of cells without treatment (% control axis "y"/. Presents mean values and SEM values for the two parallel samples.In Fig. 13 shows the inhibition of tumour growth in deprived hairline mice using ectodomain of syndecan-1.Summary of the invention
The present invention primarily relates to pharmaceutically acceptable compositions containing indecency ectodomain.In addition, the present invention relates to a method of reducing or controlling the growth of tumor cells by delivery of such sindelarova acetaminophe protein to tumor cells in the extracellular environment surrounding the cell.The methods of the present invention is applicable to both malignant and neelakantan tumor cells and they re the lymphoma or adenomas.Definition
For a more clear and consistent understanding of the specification and claims, includes the terms listed below, the following is a transcript of such terms.The growth of cells. The term "cell growth" refers to cellular replication, or the rate of cell division both in controlled and uncontrolled conditions. Therefore, the growth of cells implies the rate of cell division and replication.Cancerous. The term "malignant" refers to the uncontrolled growth of cells.More Mature (definitive) phenotype. At the mention of the cells having "more Mature phenotype" is meant that the cell has the phenotype, usually characteristic of some types of cells are more Mature than this cell. The phenotype can be determined by one or more phenotypic characteristics. So, for example, the shape of epithelial cells is a more Mature phenotype of mesenchymal-like shape; therefore, in this example, "a more Mature phenotype represents the morphology of epithelial cells, but not mesenchymal-like shape. Terminal differentiated mesenchymal cell is a "more Mature phenotype than condensing mesee filaments are indicators of a less Mature phenotype, than organized filaments.An effective amount. "Effective amount" of any agent represents the quantity of such agent, sufficient to obtain the desired result, especially when using such an agent to the animal or person. An effective amount of ectodomain of syndecan-1 in the compositions and methods of the present invention is an amount sufficient to reduce the growth of tumor cells, preferably up to speed normal growth of specific cell types.Application. The term "application" refers to the introduction of ectodomain of syndecan in accordance with the invention into the animal or human body by appropriate means known in this field of medicine, and such term includes injectable, oral, enteral, subcutaneous and parenteral (e.g. intravenous) application, but is not limited to them.The impact of ectodomains of syndecan. Under the "impact" on the cell sindelarova of ectodomain in the compositions of the invention assumes that the external environment of the cells is ensured by the quantities sindelarova of ectodomain, which is effective for the promotion of the desired salt. It is implied that the term "pharmaceutically acceptable salts" embraces salts sindelarova of ectodomain of the present invention. Such salts can be formed from pharmaceutically acceptable acids or bases, for example, such acids as sulfuric, chloride-hydrogen, nitrogen, phosphate and so on, or such grounds as hydroxides of alkaline earth metals, hydroxides of ammonium, hydroxides of alkylamine etc.Pharmaceutically acceptable composition. It is implied that the term "pharmaceutically acceptable composition" includes solvents, carriers, diluents, etc. that are used as additives in drugs sindelarova of ectodomain of the present invention to use compounds on patients (human or animal) in need of treatment. Such additives can perform certain functions, for example, to provide an appropriate ionic conditions for use, to stabilize indecency ectodomain from the decontamination or degradation and/or increase half-life sindelarova of ectodomain. Pharmaceutically acceptable compositions medically compatible with the host organism, in which the use of the medication.Practically free of natural contaminants. The material is considered to be "practically free of natural contaminants" if it has been subjected to significant clearing of substances that are normally present in it in its natural state, prior to such treatment, and such impurities usually find in the natural state of a substance in vivo or in vitro, are practically absent in the final preparation of such material. When applied to the object in need of treatment, indecency ectodomain invention contains almost no natural contaminants that are associated with sindelarova ectodomain as in vivo (in the body of the host from which emit ectodomain) and in vitro (chemical synthesis). The term "virtually absent" means that such impurities are either absent, or present in such low concentrations that their presence (1) does not prevent the desired therapeutic effect of the active agent (in this Slupi its application to the patient, in need of the treatment, and (2) does not harm to a patient using such a composition.Description of the invention
The present invention is based on the discovery of the fact that ectodomain of syndecan have some biological functions and is able to provide functions such cells in the presence on the outer surface of cells, different from those that synthesize such indecency ectodomain. Syndecan are proteins associated with the membrane. Unexpectedly, it was found that extracellular formed indecency ectodomain by itself sufficient for the reconstruction of a more differentiated morphology of tumor cells and inhibit the growth of malignant cells. In this case, an example of the invention is syndecan-1.All syndecan contain cytoplasmically domain, a transmembrane domain and extracellular domain. The extracellular domain is ectodomain. As discussed Jalkanen with TCS. in the book "the Receptor for the extracellular matrix, ed. J. MacDonald and R. Mechem, Academic Press, San Diego, PP 1-37 (1991), syndecan contain highly conserved homologous sequences in three separate areas of their ectodomains. Dibasic sequence n the location of the outer leaf of the plasma membrane and the ability to function as a protease-sensitive site, which ensures the removal of intact ectodomain from the cell surface.The core protein of the human syndecan-1 contains a 310 amino acid residues. Murine and human syndecan-1 highly structurally and functionally homologous to each other. Human syndecan-1 has the size, charge, hydrostatic density and GAG composition, identical to the corresponding characteristics of the mouse syndecan-1. Ectodomain human syndecan-1, similar to the murine analogue, binds to fibrils of collagen type 1 and fibronectin, but not able to connect with laminin or vitronectin.The sequence of the human syndecan-1 is known, and it was subjected to cloning (with Mali al., J. Biol. Chem. 265:6884-6889 (1990)).In accordance with the numbering according to Fig. 2 the article with Mali al., J. Biol. Chem. 265:6884-6889 (1990) amino acids 1-251 are ectodomain human syndecan-1 (with attached signal secretion), hydrophobic covering membrane domain contains the following 25 amino acid residues (amino acids 252-276), and cytoplasmic domain contains the last 34 amino acid residue (amino acids 277-310). The signal peptide sequence of synthesizing its cells the secretory signal is not required for suppression of tumor growth or differencirovannyh functions ectodomain invention.In this regard, the sequence of ectodomain of the present invention contains such passages sindelarova amino acid residues 1-251 that preserve sites joining the GAG and the desired function ectodomain, as is the case with, for example, ectodomain containing amino acids 1 to 251 (with the secretory signal and splitting on the RK website), 18-251 (without secretion signal, but with splitting on the RK website), 1-231 (secretion signal, but with splitting on the RR site) and 18-251 (without the secretory signal, but with splitting on the RR site). Ectodomain with carboxykinase on the website, located between amino acid residues 231-251, or a fragment of a secretion signal, a smaller fragment of the amino acids 1-17, may also be used, since it can be expected that such decisions retain the biological properties of ectodomain.Although human and mouse ectodomain identical amino acid level is only 70%, all prospective sites joining glycosaminoglycan (GAG) in mouse and human sequences are identical. Five possible sites of attachment of glycosaminoglycans and collagen of ectodomain human syndecan represent positions 37, 45, 47, 206 and 216. Two of these sites belong to karjat identical signal N-glycosylation m proteinase-sensitive dibasic RK site, neighboring extracellular outer surface transmembrane domain. Human syndecan also contains a second dibasic RR sequence of 18 residues in addition to the sequence RK. Proteolytic cleavage at this site may also lead to the formation of ectodomain of the present invention, which contains all intact GAG sites.The transmembrane domains of human and mouse syndecan-1 is identical to 96% (only difference in the human syndecan is the substitution of alanine for glycine), and cytoplasmic domains at 100% identical in mouse and human syndecan.Indecency ectodomain, such as ectodomain human syndecan, may be obtained by recombinant methods in the body of any desired host. However, preferably, but not necessarily, to use the host cell type, similar cell type of the tumor in order to provide the most similar composition GAG relative to cells in non-neoplastic condition. Available a large number of deposited cell lines, which are specific against human tissue or possess the characteristics of different cell types.So is speccie and geneticin for further breeding steadily transfection cell clones. Clones cell line S115 (see Fig. 3) Express a wild type mouse syndecan-1 (wild type), deletion mutant with a single arginine residue only in the cytoplasmic domain (tailless) or flat ectodomain of syndecan-1 (ecto). Wild type syndecan-1 and cytoplasmic deletion mutant (tailless) cloned in the EcoRI site pBGS eukaryotic expression vector. Ectodomain construct cloned in rmania vector in order to ensure effective levels of expression in the presence of the hormone, because MMT LTR promoter is induced by the same steroid hormone that and cells. Not necessary to use such a vector, because in this area there are a large number of such expression vectors. The expression of syndecan-1 on the surfaces of cells detected using monoclonal antibodies, which was exemplified by the above-mentioned mAB 218-2, which recognizes ectodomain crustal protein mouse syndecan-1, and actin filaments visualize using rhodamine-conjugated phalloidin as an indicator of status differentiation and growth of cells.In the absence of testosterone S115 cells demonstrate the presence of organized actin frame the I and acquires a spherical shape, it also suppressed the expression of syndecan-1 on the surface of cells, clones of wild-type and tailless clones expressing syndecan-1 on cell surfaces to restore the organization of actin filaments, in spite of treatment with testosterone. Because transfection Bukhvostov mutant also induced changes similar to syndecan-1 wild-type cells S115 transfections simple ectodomain and formed more than 50 independent clones secreting different levels of ectodomain in the cultural environment. The surface of such cells acquire only a weak color under the action of syndecan-1, and these cells still show a well-organized actin filaments and epitheloid morphology. These results show that ectodomain of syndecan-1 has sufficient properties to restore epithelioid morphology of S115 cells treated with testosterone to the morphological status, characteristic of a more Mature phenotype, and is a valuable anti-cancer drug.In the case of non-neoplastic cells syndecan is expressed in the epithelial cells, mesenchymal cells, pre-B cells and plasmacytic, but is not expressed on B-cells. Ani. Therefore, the method of the present invention is particularly useful in fighting tumors of epithelial, mesenchymal, pre-B and plasma cells. Mainly, the methods of the present invention are used to slow the growth of steroid-reactive tumors, especially estrogen or androgen-reactive tumors (tumors that grow better in the presence of steroids, such as estrogen or androgen), including tumors of the breast cells, tumor cells of the mucous membrane of the uterus and the tumor cells of the prostate.For treatment of humans and animals ectodomain of syndecan-1 is used in a pharmaceutically acceptable solution at dosages sufficient to restore the state normal growth of the tumor or malignant cells, the evidence of which is the slow growth rate. Singakademie pharmaceutically acceptable solution can be applied in any form, providing preventive, palliative, preventing or regressing effect on tumor growth.The number of compositions of ectodomain of syndecan-1 of the present invention, which is applied on the patient and the duration of such use, may be determined by monitoring apohelion of the present invention is preferably applied on the tumor cell target when extracellular concentrations of 0.7-1.0 nm (see Fig. 11), but can be any concentration sufficient to reduce tumor growth. Ectodomain can be used either locally (as is the case with concentrated delivery directly to the target organ) or systemically (for example, by prior rate through the bloodstream). Dose of syndecan used on the patient (both human and animal), should therefore be chosen with regard to the volume (for example, blood volume), which apply ectodomain, and the type of tumor, which is the target for the drug.For example, if you want a permanent effect sindelarova of ectodomain requires more frequent reception of the dosage of medicine than for the case when you have only a temporary impact sindelarova of ectodomain on the tumor. So, for example, 1 nm sindelarova of ectodomain containing amino acids 1-251, corresponds to a concentration of 0.2 mg/l (200 mg/l) in the blood or for local use in the area. Typical dosing system sindelarova of ectodomain used in the methods of the present invention for the treatment of humans or animals, include the quantity, ensure the final concentration in the blood, most preferably, 0.2 mg sindelarova Ecodom liters of blood. However, since it is assumed that the effects sindelarova of ectodomain are local (for example, effects on specific cell membrane), isolated and kinetically restrictive, in theory a minimum dosage may be higher than specified to achieve a beneficial therapeutic effects.Indecency ectodomain can be used in any way that ensures the delivery of effective amounts of drug to the desired active area, for example by injection. In the case of parenteral administration preparations containing indecency ectodomain can be used on the patient in need of such treatment in combination with a pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions and emulsions, examples of nonaqueous solvents can serve as propylene glycol, polyethylene glycol, vegetable oil, fish oil and suitable for injection complex organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffer parenteral medical media, including sodium chloride, dextrose ringer's solution, dextrose and chloride is e and nutritional additives, electrolytic additives, for example additives on the basis of dextrose ringer, etc.Medication containing indecency ectodomain (pharmaceutically acceptable solution containing therapeutically active indecency ectodomain), can be used with catheters or pumps, especially in those cases when it is desirable to ensure the delivery of ectodomain in localized high concentrations. Medication containing ectodomain of syndecan-1, can be applied subcutaneously or directly into the soft tissue with implanted devices, inert in relation to the liquid environment of the body. Such devices and implant system known in the field. For example, a ceramic system for delivery of proteins described in WO 92/00109.Medication containing ectodomain of syndecan-1, can be applied with the use of such molecules as part of the chimeric molecule (or complex) designed to act on specific target organ, for example as part of antibodies, which recognize determinants of the target tissue, organ or cells in neoplastic and non-neoplastic condition.Pharmaceutically acceptable solution containing ectodomain of syndecan-1, can be used the drugs for cancer, optimal application sandcasters compositions of the invention are particularly useful in this case.Topical application is preferably carried out using one of two methods. According to the first one therapeutically active indecency ectodomain can be mixed with suitable pharmaceutically acceptable carriers and, optionally, with penetration enhancers to facilitate delivery of the active agent through the skin with the formation of ointments, emulsions, lotions, solutions, creams, gels, etc. and then the mixture is applied on a particular area of skin. According to the second method of therapeutically active indecency ectodomain may be injected into the patch or transdermal delivery, in accordance with known technology of preparation of such patches and delivery systems.The use of medications in the form of prolonged selection more convenient for the patient when required repeated injections over a long period of time or when it is desirable lasting impact ectodomain to a tumor cell. When used as an intravenous dosage form of the composition of the present invention demonstrate a rather fast start on lituano directly on the cell, if it is associated with tissue or organ of the body, or use can be system, in an environment where there is such a cell, for example in blood or cerebrospinal fluid. The system application on the patient's body, for example the use of the drug through the bloodstream, facilitates the treatment of patients whose tumor cells are found in more than one area of the body.Ensuring sindelarova of ectodomain in the form of product design on the basis of expression sindelarova of ectodomain, which secretes ectodomain in effective quantities, also seen as "application". So, for example, through the use of hematoencephalic barrier can be achieved using known viral vector systems for the delivery of DNA sindelarova of ectodomain providing expression ectodomain and its secretion into the extracellular environment, as is the case, for example, retroviral systems described in WO 93/03743, WO 090/09441, and articles Breakefield, X. A. al. The New Biologist 3:203-218 (1991) and Huang, Q. al., Exp. Neurol. 115:303-315 (1992).Pharmaceutically acceptable composition of the invention containing ectodomain of syndecan-1, get a known manner, for example by conventional mixing, RA is W ill result application due to its ability to slow or prevent tumor growth or relapse and the ability to change the genotype of the cells on the genotype of a more differentiated state as on the people, and on animals. In accordance with the present invention the composition of ectodomain sindikata-1 use their own mechanisms of the body for promotion of the differentiation of specific cell types to their maximum potential.It is implied that the compositions and methods of the invention are not limited to using only syndecan-1. It is known that syndecan-1, syndecan-2, syndecan-3 and syndecan-4 contain similar domain structure. It is known that the differentiation of some cell types associated with the loss of syndecan-1, but with the advent of another member sindelarova family (Bernfield, O., al., Annu. Rev. Cell Biol., 8:365-393)).For example, in the case where the bronchial epithelium forms sprouts, pulmonary mesenchyme loses syndecan-1, but becomes syndecan-2. In tumors from cell types that lose syndecan-1 during differentiation, but Express another syndecan, we can expect the use of ectodomain of syndecan, which is expressed in a state of differentiation.The examples below are merely illustrative and do not limit the scope of the invention.EXAMPLES
The following examples illustrate but do not limit the image is osanai transfection and subsequent selection steadily transfection cell clones in the presence of geneticin, as described Leppa with TCS. , Proc. Natl. Acad. Sci. USA. 89:932 (1992), received the clones cell line S115 (see Fig. 3), which expressed wild type mouse syndecan-1 (wild type), deletion mutant with a single arginine residue only in the cytoplasmic domain (tailless) or only ectodomain of syndecan-1 (Ecto 2; see Fig.3). Three such forms and their respective owners designed as follows.cDNA mouse syndecan-1 full length described in the article with Mali al., J. Biol. Chem. 268:24215-24222 (1993), cloned in the EcoRI site of Bluescript SK+ (Promega).1) EcoRI insert design Bluescript cloned in the EcoRI site of the vector pBGS (Mali from al. , J. Biol. Chem. 268:24215-24222(1993)) and confirmed the orientation. This construct was designated as "wild type".Mutagenic oligonucleotide of 25 bases, having the sequence: 5' G CTG TAC CGC TAG CAG AAG GAC-3' [SEQ ID N:1] that contains the stop codon and Nhel restriction site (underlined) was used to convert the codon for the second amino acid (methionine) of the cytoplasmic domain, following the transmembrane domain, in the stop-codon. The mutation was confirmed by restriction digestion and dideoxy sequencing. EcoRI insert design Bluescript cloned in the EcoRI site amplifierarava pBGS vector (alaemon cytoplasmic domain, meant as a "tailless".Mutagenic oligonucleotide from 33 basis:
5'-GACACCTCCCAGTACTCACTTCCTGTCCAAAAG-3' [SEQ ID N:2], containing a stop codon, and Scal-site (underlined) was used for transformation of the first codon (E) after site ectodomain sensitive to the dibasic protease, in the stop-codon. The mutation was confirmed by restriction digestion and dideoxy sequencing. It was found that the analyzed object is a Bluescript-ecoconstruction. EcoRI insert Bluescript-ecoconstruction cloned in the EcoRI site of the vector pJC119R (with Miettinen al., J. Cell Sci. 107: in press, (1994)). Xhol-digested ecto-box of pJC119R-ecoconstruction ligated into the Xhol site pMAMneo eukaryotes transfection vector obtained from Conteh, Palo Alto (Leppa with al., Proc. Natl. Acad. Sci. USA. 89, 932(1992)), and the orientation was confirmed by restriction perevarivaniya.Example 2
Expression of mutant syndecan-1, normalizing malignant growth in S115 cells
Syndecan-1 wild-type and cytoplasmic deletion mutant (tailless) cloned in the EcoRI site pBGS eukaryotic expression vector (Mali from al. , J. Biol. Chem. 268:24215 (1993), and ectodomain design has cloned into the pMAMneo vector in order to achieve an effective level, the system was not suppressed by testosterone. The expression of syndecan-1 on the surfaces of cells were detected using mAb 281-2 (Jalkanen with al., J. Cell Biol. 101:976 (1985), which recognizes ectodomain crustal protein mouse sinicina-1, and actin filaments were visualized using rhodamine-conjugated phalloidin.Cells (S115+, wild-type, tailless and Ecto 2) were cultured for four days on slides in DMEM-5%FCS-lM Na pyruvate in the presence of 10 nm testosterone, with the exception of S115 cells that were cultured without testosterone in the environment DMEM-4%DCC-FCS (charcoal, which caused gastrin, provides the removal of serum endogenous steroids) in the presence of 1 mm Na pyruvate (pyramidographia sodium - approx. trans.) the Cells were fixed with 0.1% of Triton-X-100, 2% paraformaldehyde, and incubated in the presence of rhodamine-conjugated phalloidin (Sigma). The expression of syndecan-1 on the cell surface was visualized by incubation of living cells for 1 hour on ice in the presence of rat mAb 281-2 (discriminating ectodomain mouse syndecan-1); then they were fixed with 2% paraformaldehyde and bound mAb 281-2 visualized using FITC-conjugated rabbit Anticriminal Ig is for these cells in epitheliale condition. In the presence of
hormone actin disarmed and acquires saricoban form and observed suppression of expression of syndecan-1 on the cell surface, as it was shown earlier Leppa with TCS. Cell Reg. 2, 1 (1991), Fig.4.Clones of wild-type and tailless clones expressing syndecan-1 on the cell surface, restore the organization of actin filaments in spite of treatment with testosterone, Fig. 4.Example 3
The influence of secreted ectodomain of syndecan-1 on cultured S115 cells
Because transfection of tailless mutant causes the same changes as in the case of syndecan-1 wild-type cells S115 was transfectional using ectodomain. Received more than 50 independent clones secreting different number of ectodomain in the culture medium (see Fig. 5, 6 and 7). The surface of such cells are only weakly stained by syndecans-1, but they still showed organized actin filaments and epitheloid morphology (Fig. 4). These results confirm the fact that ectodomain of syndecan-1 has a sufficient set of properties for the reconstruction epithelioid morphology treated with testosterone S115 cells.With the purpose of detailed analysis POS cultural environment using mAb 281-2 to ectodomain crustal protein of syndecan-1. Two separate steadily transfection cell clone secreting high amounts of syndecan-1 in the culture medium (Ecto 2 and Ecto 23) and two cell clone with low expression (Ecto 15 and Ecto 34) was chosen for further analysis (Fig.5).A clear correlation between the expression of ectodomain of syndecan-1 and reorganization of actin filaments was observed in the presence of 10 nm testosterone: Ecto 15 and Ecto 34 with low expression of syndecan - 1 contained disorganized, mainly globular actin, whereas Ecto 2 and Ecto 23 clones expressing ectodomain of syndecan-1, showed epithelioid morphology with organized bundles of actin filaments (Fig.6). As shown previously, increased expression of intact syndecan-1 suppresses tumor growth of treated with testosterone S115 cells (Leppa with al., see the above-cited reference) and in this case, the clones Ecto 2 and Ecto 23 with high expression of ectodomain of syndecan-1 also demonstrate limited growth on soft agar. However, the clones Ecto 15 and Ecto 34 with low expression of ectodomain of syndecan-1 demonstrate growth on soft agar, typical for related S115 cells (Fig.7). Experiment on soft agar shows that in addition to ptx2">Example 4
Isolation and purification of sindelarova of ectodomain from Ecto cell cultures
Because ectodomain of syndecan-1, seems to be responsible for the suppression of malignant growth in androgen-treated cells S115, collected the conditioned medium from Ecto cell cultures to highlight ectodomain. Air-conditioned cell culture medium was denaturiruet with 2 M urea and boiled before loading DEAE-cefaclor column, was added 50 mm Na acetate (pH=4.5) and Wednesday froze to +4oC. the Column was washed with 0.2 M NaCl, 2M urea, 50 mm Na acetate (pH=4.5) and the bound material was suirable using 1 M NaCl, 2 M urea, 50 mm Na acetate (pH=4.5). The fractions containing ectodomain of syndecan-1 was subjected to dialysis countercurrent to phosphate buffered saline (PBS) at 4oC. the Number of ectodomain of syndecan-1 in the fractions was determined by slot blot and subsequent enhanced chemiluminescence, using mAb 281-2 (Example 2 and Miettinen H.M. with al., J. Cell. Sci. in press (1994)) and compared this number with a known standard syndecans-1.Ectodomain of syndecan-1 of culturebound environment Ecto cells biochemically similar ectodomain of syndecan-1, the s in the preparation of syndecan-1 and the drug was tested on hormone-treated cells S115. As shown in Fig. 8, such low concentrations D-selected ectodomain of syndecan-1 as 1 nm inhibit the growth treated with testosterone S115 cells (Fig. 8). The same concentration only slightly inhibits the growth of NMuMG cells, which were used as normal epithelial cells (Fig. 8). Ectodomain of syndecan-1 was also isolated from the cultural environment NMuMG cells and again 1 nm concentration of this drug inhibited the growth of processed hormone S115 cells (Fig. 9). Processing DEAE-selected ectodomain heparinase completely inhibited religiuous activity of such preparations (Fig. 9), which involves the failure to involve in this process the crustal protein of syndecan-1.DEAE-selected ectodomain of syndecan-1 was further purified using mAb 281-2 immunoaffinity column: DEAE-selected ectodomain of syndecan-1 in PBS was loaded into the mAb 281-2 Sepharose CL-4B immunoaffinity column in accordance with the methodology described Jalkanen with al., J. Cell. Biol. 105: 3087 (1987) and the bound material was suirable 50 mm triethylamine (pH=11,5). The fractions containing ectodomain of syndecan-1, dialyzed countercurrent to the distilled water and then liofilizirovanny. After that ectodomain of syndecan-1 suspended in DMEM. (Killed the NGO-purified ectodomain of syndecan-1 was observed inhibition of growth treated with testosterone S115 cells and only a small effect was achieved in NMuMG cells (Fig.10). On the other hand, heparin sulfate (HS) or chondroitin sulfate (CS) glycosaminoglycans chains themselves do not inhibit the growth of cells S115, even if they are used with concentrations thousands of times greater than the concentration of ectodomain of syndecan-1 (Fig.11).Example 5
The influence of selected ectodomain of syndecan-1 on cultured cell lines
The inhibitory effect of selected ectodomain of syndecan-1 was also tested in several other cell lines. Such objects include poorly differentiated carcinoma cells squamous cells (Carb), the tumor cells are breast cancer human (MCF-7; ATS NTV 22), S115 cells containing (S115+) and do not contain hormone (S115-), NIH T fibroblasts (ATSS CRL 1658), normal epithelial cells of the mammary gland (NMuMG; ATS CRL-1636), and human keratinocyte cells (Nasat; Fig. 12).Cells were cultured and analyzed, as shown in Fig.8, in the following environments within specified time periods: CarB cells (M. Quintanilla, K. Brown, M. Ramsden, A. Balmain, Nature 322, 78 (1991)) were cultured in HAM-F12-10% FCS for four days; cells MCF-7 were cultured in DMEM-5% FCS, supplemented with 10 nm estradiol (E2) and 10 μg/ml insulin for 4 days; cells S115+ and S115 - cultivated, as pakvali in 10% FCS-DMEM for 4 days. Because of S115 cells grow much slower than cells S115+, 3000 cells S115- (for other cell lines 1500 cells) proportionally added to the well in order to obtain results comparable with those obtained for cells S115+. In this regard, in the case of cells S115 - 3000 cells were transfectional on the plate in contrast to 1500 cells for other samples.Line of cells that form tumors (CarB, MCF-7, S115+), showed strong growth inhibition when exposed to ectodomain of syndecan-1 at a concentration of 1 nm (Fig.12). In contrast, there has been only a slight inhibition or lack thereof in the case of the other tested cell lines (S115-, NIH T, NMuMG, HaCaT; Fig.12), which in all respects are non-carcinogenic. The effects of hormone doubles the growth rate of cells S115 (Leppa with TCS. see the link cited above), however, if the culture impose ectodomain of syndecan-1, the growth of S115 cells in the absence of androgen is 5.4 times faster than the growth of these cells S115 in the presence of testosterone (Fig.12). This result is associated with inhibition of malignant behavior of cells S115+ and undisturbed growth epitheloid S115 cells.Example 6
Suppression of tumor growth in vivo using ectodomain Sindh is NK mouse syndecan-1 full length cloned in Bluescript SK+ vector (10) and mutagenic 33-base oligonucleotide 5'- GACACCTCCCAGTACTCACTTCCTGTCCAAAAG-3' [SEQ ID N:2), containing a stop codon (Bold) and Sal website (CAGTAC), with the aim of turning the first amino acid (s), the following acetaminophe site, sensitive to the dibasic protease, in the stop-codon. Mutation selectively restrictive digestion and confirmed dideoxy sequencing. Syndecan-1 wild-type and cytoplasmic deletion mutant has cloned in the EcoRI site pBGS eukaryotes expression vector (Mali from al., J. Biol. Chem. (1993) 268, 24215-24222). Ecto mutant ligated into the Xhol site pMAMneo eukaryotic transfection vector /Leppa with al., PNAS (1992) 89, 932-936), because it was known that pMAMneo transfection S115 cells work well in a bioreactor system (personal message. Sari Ala-Uotila, Turkish center for biotechnology). S115 cells were transfectional using the liposomal transfection and subsequent selection using Geneticin, as described above (Leppa with al., PNAS (1992) 89, 932-936).Cells S115 and transfection cell clones were cultured in DMEM-5% FBS-1 mm Na pyruvate in the presence of 10 nm testosterone, with the exception of S115 cells which were cultured without testosterone in DMEM with 4% DCC-PBS (metallov of serum) in the presence of 1 mm Na pyruvate.For tumor growth subconfluent culture were separated with trypsin, washed with DMEM and counted their number using the counter of Coulteri (Coulter electronics). Cells resuspendable in DMEM at a density of 107/ml and kept on ice until injection. Itemnum (Athymic) male mice devoid of hair (nu/nu BALB/cABom) aged 6-8 weeks (Bocholter, Ry, Denmark) did subcutaneous injection of 0.2 ml of cell suspension. Simultaneously implanted vladicescu testosterone capsule. Mice were regularly observed the development of the tumor and the size of tumors was measured after a certain period of time in two perpendicular directions. After conciliation animal lungs and liver were investigated on the possible appearance of metastasis. The size of tumors was measured after 6, 11 and 15 days after injection and were applied to the graph shown in Fig.13, the average values for the five individual tumors. Transfection ectodomain cells gave only the acute inflammatory response and did not show tumor growth in contrast to wild-type cells, which formed rapidly growing tumors. This experiment demonstrates the effectiveness of ectodomain of syndecan-1 as an agent that suppresses tumor in vivo.
< / BR>where X is O,
< / BR>R1indicatesor< / BR>R2and R3each, independently of one another, denotes H, A or benzyl;
A denotes alkyl with 1-6 C-atoms;
D denotes amidinopropane, aminomethyl, aminohydrocinnamic, 5-methyl-1,2,4-oxadiazolidine-3-yl or guanidinate;
r and s independently of one another denote 0, 1, 2, 3 or 4;
however, if necessary, free amino - or amidinopropane can be protected partially or fully protective for the amino function groups, as well as their enantiomers, diastereomers and physiologically acceptable salts
FIELD: medicine, pediatrics.
SUBSTANCE: the present innovation deals with treating motor-autonomic disorders in children associated with affected function of central nervous system. For this purpose one should puncture perineural areas in the region of the main nervous trunks with alfetin dissolved in cerebrolysine. Additionally, one should puncture in projection area of cervical and lumbar spinal thickenings and areas that correspond to segmentary innervation of organs with affected function and, also, in scalp areas depending upon the character of patient's disorders. The method suggested provides improved autonomic-trophic impact of nervous system.
EFFECT: higher efficiency of therapy.