Pharmaceutical composition containing c-kit-ligand and hematopoietic factor, a method of increasing levels of stem cells in the peripheral blood, the antagonist of c-kit-ligand, the antisense nucleic acid molecule, the method of increasing levels of peripheral blood cells ex vivo

 

(57) Abstract:

The invention relates to the field of medicine and relates to pharmaceutical compositions containing the c-kit-ligand and hematopoietic factor, a method of increasing levels of stem cells in the peripheral blood, an antagonist of c-kit ligand, antisense molecules of nucleic acid, the method of increasing levels of peripheral blood cells ex vivo. The invention includes a pharmaceutical composition on the basis of c-kit-ligand, representing the polypeptide encoded by the nucleic acid SEQ ID NO:l, or the naturally occurring hematopoietic variant polypeptide and an effective amount of hematopoietic factor, a method of increasing the level of stem cells in the peripheral blood of a mammal using the specified composition. In addition, the disclosed antisense nucleic acid for use as therapeutic agents, as well as a way to increase levels of peripheral blood cells ex vivo, comprising the processing of peripheral blood cells ex-vivo specified composition. The advantage of the invention is the creation of new homeopathic remedies. 4 C. and 10 C.p. f-crystals, 35 ill., 3 table.

The invention is the as links.

Work on this invention was carried out in accordance with the agreement on subsidies ROI-CA 32926 and ACS MV 246D from the national institutes of health and American cancer society, respectively. The U.S. government has certain rights in this invention.

Prior art

Various publications are referenced in this application, marked with Arabic numerals in parentheses. A full bibliography is given at the end of the description. For a more complete description of prior art prior to the present invention, the disclosure of these publications in their entirety is entered into this application by reference.

Protooncogene c-kit encodes a transmembrane receptor tyrosine kinase for an unidentified ligand and is a member of the subfamily kit receptor, the receptor for colony stimulating factor - I(CSF-I) a growth factor, derived from platelets (PDGF) [7, 41, 57, 3]. Recently, it was shown that C-kit is the allele at the locus of white spot (W) (White spotting) mouse [9, 17, 35]. Mutations at the W locus affect proliferation and/or migration, and differentiation of germ cells, pigment cells and different populations of hematopoietic cells systemsthe on-line differentiation of erythroid and mast cells, as well as stem cells, which leads to macrocytic anemia, which is lethal for the homozygotes for the most powerful of the W allele [46], and to the complete absence of fat cells in the connective tissue and mucous membrane [72]. W-mutations act in a cell Autonomous manner [28, 46], and in accordance with this property W-mutation was shown that the mRNA of C-kit is expressed in the targets of W mutations [35] . High levels of c-kit mRNA was detected in embryonic fat cells originating from bone marrow, and the lines of the fat cells. Somewhat lower levels are found in melanocytes and erythroid cell lines.

Identification of the ligand for c-kit is of great interest for professionals, because it can have pleiotropic effects on various cell types, which Express c-kit and which are exposed to W-mutations in vivo. On the basis of knowledge about the function of c-kit/W can be obtained important information about the types of cells that can produce c-kit-ligand. The absence of mast cells in connective tissue, and mucous membrane of the gastrointestinal tract W/Wvmice indicates a role of c-kit in the development of fat cells. Fat cells derived from the bone marrow (VMMS), ablauts the but-intestinal tract (MMS) [92, 93]. Mast cells of connective tissue originating from the abdominal cavity (STMS), in vitro necessary for proliferation of IL-4 and IL-4 [79, 75]. Interleukins IL-3 and IL-4 are characterized as hematopoietic growth factors, which are produced by activated T-cells and activated mast cells[92, 94, 95, 96, 97]. It has been suggested that there is, in addition, the growth factor of the fat cells, which is produced by fibroblasts [47]. In the absence of IL-3 BMMC and STMS obtained from the abdominal cavity, can be supported by the joint cultivation with T-fibroblasts [98]. However, BMMC, originating from W/Wvmice, and from mice homozygous for a number of other W-alleles, were incapable of proliferation in the systems of co-culture with fibroblasts in the absence of IL-3 [99, 100, 38]. It is possible to make assumptions about a specific function of the receptor c-kit in Mature fat cells and come to the conclusion that the ligand of c-kit receptor is produced by fibroblasts. Recently Huff and his colleagues report was made stimulation colonies fat cells derived from lymph nodes of mice infected with the nematode Nippostronglyus brasiliensis using the concentrated conditioned medium for fibroblasts is oneiromancy activity (denoted KL), which in the absence of IL-3 promotes the proliferation W/WvBMMC. In addition, it was shown that KL contributes to the formation of the hearth differentiation of early progenitor cells erythroid series (burst-fire) (BFU-E). Biological properties of KL correspond to the intended properties of the c-kit-ligand in relation to the biology of fat cells and erythropoiesis. Defective W-mutations are cell-Autonomous, and in accordance with this property was illustrated by the expression of c-kit-PHK in target cells W-mutations [35, 39]. Recent characterization of molecular damage of several mutant alleles showed that they are mutations with loss of function, resulting in disruption of normal activity or expression of c-kit-receptor[35, 100, 101, 36].

Mutations in the steel locus (SI) of mouse chromosome 10 lead to the emergence of phenotypic characteristics that are very similar to the symptoms observed in mice bearing W mutations, i.e., they affect hematopoiesis, hematogenous and melanogenesis [5, 47, 51]. Many alleles at the SI locus are known (they are predominantly mutations), and different alleles vary greatly both in terms of its impact on the line of differentiation of various cells, and the extent of their dominantely cells, deprived of the coating pigment, and die from perinatal makrotsitarnoy anemia [5, 50]. Mouse homozygous for the SI allele, although they are viable, but they have severe macrocytic anemia, lack of pigment in the skin and sterility. Heterozygotes for SII+and SId/+ - alleles have pale color of the skin and moderate macrocytic anemia, however, they are able to produce offspring, but their gonads reduced in size. In contrast to the W-mutations SI-mutations do not have a cell autonomy, and, apparently caused by a defect in the microenvironment of the target for the specified mutations [28, 30, 12]. On the basis of parallel and additional characteristics of mice bearing SI and W mutations, the authors of this application and others expressed the provisional hypothesis that the gene product is a ligand of c-kit-receptor [51, 9].

Protooncogene c-kit is a normal cellular equivalent of the oncogene v-kit HZ4-virus sakray cats [7]. c-kit encodes a transmembrane receptor tyrosine kinase that is a member of the subfamily of receptors for growth factor derived from platelets, and is a gene product of the locus of white spot[9, 17, 23, 35, 41, 57]. The identity of c-kit with W-locus witness the Genesis, and hematopoiesis during embryonic development and in the Mature animal [47, 51]. Confirmation of the above hypotheses regarding the functions of the c-kit receptor is the fact that c-kit mRNA is expressed in the target cells for W-mutations[3, 24, 25, 35, 39].

Based on known data about the function of c-kit/W in the fat cells has recently been identified and characterized the ligand of c-kit receptor (KL) [2, 14, 37, 38, 56, 58, 59]. In accordance with the predicted functions of the c-kit receptor in geobase KL stimulates the proliferation of mast cells derived from bone marrow and connective tissue; and erythropoiesis KL in combination with erythropoietin stimulates the formation of foci of differentiation of early progenitor cells erythroid series (7-14-day BFU-E). In addition, a recent in vitro experiments with KL demonstrated increased proliferation and differentiation of erythroid, myeloid and lymphoid precursors in the case when KL used in combination with erythropoietin, GM-CSF, G-CSF and IL-7, respectively, suggesting that c-kit receptor plays a role in the formation of precursors of several hematopoietic cell lines differentiation [27, 37].

Mutations in steel-locus mississi W-mutation i.e. they affect hematopoiesis, gametogenesis, and melanogenesis [5, 47, 51]. Recently, it was shown that the ligand of c-kit-receptor KL is allelic with mouse-steel-locus, as evidenced by the set of observations the fact of delegation in SI-sequences from several strong SI-alleles [11, 38, 59] . According to the ligand-receptor relationship between KL and c-kit SI-mutations affect the same cellular targets, as W-mutations, however, in contrast to the W-mutations SI-mutations are not cell-Autonomous and influence the microenvironment of the c-kit receptor [12, 28, 30]. Mutations in steel-locus are predominantly mutations, and different alleles vary widely both in terms of its impact on the line of differentiation of various cells, and the extent of their dominance [47, 51]. The original allele SI is an example of a strong SI-mutations. SI/SI-homozygotes lack germ cells, deprived of wool dyeing and already die in utero from macrocytic anemia [5, 50] . Mouse homozygous for SId-allele, although they are viable, but they have severe macrocytic anemia, lack of wool dyeing and sterility [6]. Heterozygotes for SI/+ / SI/d/+ - alleles have brighter coloration of wool and moderate macuser blot analysis Sid/+ - DNA conducted using KL-DNA as a probe, testified EcoRI-polymorphism, which suggests that this mutation leads to deletions, point mutations or DNA-rearrangeable KL-gene (II).

Brief description of the invention

Was manufactured pharmaceutical composition, which includes c-kit-ligand (KL), purified by the authors of this application or produced recombinant methods developed by the applicants, in combination with other hematopoietic factors, and a pharmaceutically acceptable carrier. Were also developed methods for the treatment of patients, which consists in the introduction of these patients, the pharmaceutical compositions of the present invention. The present invention relates to integrated treatment with c-kit-ligand (KL) and purified polypeptide c-kit-ligand (KL) or its soluble fragment, and other hematopoietic factors. The present invention also relates to methods and compositions for ex vivo use KL both separately and in combination therapy. In this application, also described mutated antagonist KL. This antagonist can also be a small molecule. In addition describes antisense is Asano, that KL plays an important role in germ cells, mast cells and melanocytes.

The present invention relates to nucleic acid.

The present invention relates to a nucleic acid that encodes the amino acid sequence corresponding to the c-kit-ligand (KL) and a purified polypeptide c-kit-ligand (KL).

Brief description of drawings

Fig. 1. Proliferative response +/+ and W/W-BMCC conditioned by fibroblasts environment and II -3. Fat cells, derived from +/+ or W/Wv-bone marrow were cultured in the presence of 1% ZSM, 10% FCM (noncentrifugal), or in only one environment. The inclusion of 3H-thymidine was assessed after 24-30 hours of cultivation.

Fig.2. Chromatographic profiles of cleaning KL.

A. Gel-chromatography on Ultrogel ACA 54. Optical Vlastnosti at 280 nm is shown by a dotted line, and the biological activity shown by the solid line. Position when the elution of the taps of the size of the protein shown in KD.

Century Anion-exchange FPLC (liquid Express chromatography on a column of DEAE-5PW. NaCl-gradient shown by the dotted line.

C. Division prepreparation C18-column. The gradient of I-propanol is shown in dashed lines.

A. Analysis of 0.5 ml fractions from the analytical C18-column, elyuirovaniya ammonialyase buffer and a gradient of 1-propanol.

C. Analysis of 0.5 ml fractions from the analytical C4 column, elyuirovaniya water with 0.1% TFA and in the absence of 2-mercapto-ethanol.

Fig. 4. Proliferation Wv-mutant mast cells in response to KI. Fat cells were obtained from the liver of the embryo, taken from W/+W/+hybrid, either from the bone marrow of wild-type, W and W41-heterozygotes and homozygotes. The degree of proliferation of mutant mast cells was evaluated by increasing concentrations of KI in the analysis on the proliferation. Homozygous mutant mast cells shown by the solid line, heterozygous mutant mast cells - dashed line, and the fat cells of a wild type by a dotted border except W, where normal embryos can be either + / + or W/+.

Fig. 5. Comparison of c-kit expression and sensitivity to growth factor in VMS and peritoneally fat cells (STMS/PMC).

A. Fluorescent staining heparin-glikoproteid in the treated RMS and VMS using arbeidslivet.

C. Evaluation Express the requirement of antibodies to c-kit. Serum against c-kit, shown by the solid line and the non-immune control serum is shown in dashed lines.

C. assessment of the proliferative capacity of RMS in the presence of KL. 5000 cells were seeded in 0.5 ml RPMI-C-environment in the presence of 100 units/ml KL, 10% Wehi-3 CM, or one medium and two weeks to determine the number of viable cells.

Fig. 6. Assessment of the "burst" stimulating activities KL. Cells in the bone marrow and spleen were cultured in the presence of EPO (2 units/ml) and with addition of pure KL at the specified concentration. On the 7th day of cultivation to determine the number of BFU-E. the Obtained data were expressed as averages of two separate experiments, each conducted in two duplicates for each concentration KL.

Fig. 7. Assessment KL-dependent formation of BFU-E from liver W/W of the embryo. Embryos from the cross W/+ animals were collected by 16.5 day of pregnancy. One fruit of four was W/W-homozygous. Liver cells were seeded with a density of 105CL/VL in medium containing IL-3 (50 units/ml) or KL (2.5 mg/ml) or control medium. All cultures contained EPO (2 units /ml). The data obtained were expressed as the number of BFU-E/liver and represented the average of two duplicate is Ali liver.

Fig. 8. N-terminal amino acid sequence of KL and the output of the corresponding nukleinovokisly sequence using PCR. Top row: N-terminal amino acid sequence (residue 10-36) KL. Middle row: nucleotide sequences of three cDNA obtained by cloning 101 p. O. PCR product (see Fig.10) in M13 and subsequent sequence determination. Bottom line: the degenerate sequence and antisense primers used for synthesis of first strands of cDNA for PCR. This amino acid sequence presented as SEQ ID 2.

Fig. 9. Northern-blot analysis using PCR-generated oligonucleotide probes corresponding to the selected polypeptide c-kit-ligand. Using labeled probes were selected 6,5 to mRNA.

Fig. 10. The obtained cDNA corresponding to the N-terminal amino acids 10-26 KL using RT-PCR. One microgram row(A)+RNA from Balb/C-T cells was used as template for cDNA synthesis and subsequent PCR amplification in combination with two degenerate oligonucleotide primers. In Fig.10 shows the electrophoretic analysis 101 p. O. PCR product in agarose.

Fig. 11. Nucleotide successively the sequence of the long open reading frame (top) and the nucleotide sequence is shown using the single letter code for amino acids. The numbers on the right refer to the amino acids, while methionine nucleotides 16-18) is 1. Potential N-terminal signal sequence (SP) and transmembrane domain (TMS) are shown above the dotted lines, and cysteine residues in the extracellular domain circled circles. The predicted structure of the protein is shown schematically below. There are specified sites for N-glycosylation and the location of the N-terminal peptide sequence (Pep. Seg). Nukleinovokisly sequence presented as SEQ ID 1.

Fig. 12. Identification KL-specific mRNA in RNA cells Balb/c-T using Northern blot analysis. ly(A)+-PHK (4 μg) was isolated from Balb/c-T cells by electrophoresis, then transferred to nitrocellulose, and hybridisable with32P-labeled cDNA (1,4 kb) KL. In Fig. 12 shows ignacia 18S - and 28S-ribosomal RNA.

Fig.13. LTO-SDS page analysis of KL.

A. Staining KL silver.

C. an Autoradiograph125I-KL.

Fig. 14. Binding125I-K with fat cells and c-kit expressing the 2-cells.

A. IY 2/ c-kit-cells containing the vector (pLJ) expression of c-kit, and expressing high levels of c-kit protein.

C. Fat cells originating from the bone marrow what about the litter (W/+ or +/+).

Fig. 15. Joint deposition and cross-linking 125I-KI c-kit receptor on fat cells.

A. Joint deposition KL with normal rabbit serum (NRS) or two rabbit antisera against c-kit (c-kit).

C. Cross-linking KL for c-kit with disuccinimidyl substrate. LTO-SDS page analysis was performed either 12% or 7.5% polyacrylamide gel. Cross-linked samples were marked "KL+cK".

Fig. 16. RFL R-analysis TagI - digested DNA from SI/+ and II I-mice. SI-allele from mice Snew/Fej-a/and CaJ I Hm was introduced in 57L/6J-background and evaluated the progeny obtained by crossing C57BL/6J SIC3HSIC3H< / BR>
A. Hybridization KL-cDNA probe (1,4 KB) DNA from two nanamica mice (tracks SII+) and two anemic mice (tracks SIISI). It was registered the absence of hybridization with DNA from SII SI-mice.

C. Hybridization of the same blot with TIS Dra/Sal probe, which is closely related to SI (see below for a detailed description of the invention). This probe identifies Snew/FeJ - allele (4 KB) and C57B1/6J allele (2 KB) and SIc3H+SIc3Hthe homozygotes.

Fig. 17. Nucleotide and predicted amino acid sequence of cDNA KL-2 and KL-SId. Shows the nucleotide sequence of KL cDNA obtained is NC, KL-I, KL-2 and KL-SFdwas subclinically and subjected to sequencing. Where there's no shading triangles indicate the 5'- and 3'-exon boundaries, splanirovano in KL-2; and the shaded triangles indicate points of deletion in SId-cDNA. Sequence-insert SIdcDNA of 67 nucleotides shown above is a KL-cDNA sequence. The arrows indicate the putative proteolytic sites of the enzyme in the extracellular region KL-1. Signal peptide (SP) and transmembrane segment (TMS) shown by the lines above.

Fig. 18. Panel a and B. Identification by RT-PCR cloning KL cDNA from normal tissues and SId-mutant fibroblasts. Total RNA was obtained from various tissues C57B16/J mice and SId/+ fibroblasts. RT-PCR reactions with RNA (10 μg) from normal tissues and Balb/3T3 cells was performed using primers 1 and 2, and reaction with RNA from +/+ and SId/+ fibroblasts was performed using combinations of primers 1 + 2, 1 + 3 and 1 + 4. The reaction products were analyzed by electrophoresis in 1% agarose Nusieve gel in the presence of 0.25 μg/ml of ethidiumbromid. Shown ignacia # H-Nai III-DNA markers.

Fig.19. The topology of the various KL-terminal signal peptides, Sacher is Tierno lines indicate the boundaries of alternative splanirovano exon and specify the appropriate number of amino acids. Arrows indicate putative sites of proteolytic cleavage. The shaded region at the C-end of KL-SIdindicates amino acids that are not encoded KL. KL-S is the soluble form of KL produced by proteolytic cleavage or by mutations KL by C-terminal truncation.

Fig. 20. Identification of KL-1 and KL-2 transcripts in various tissues by analyzing protection from RNase32P-labeled antisense ribsand (625 Amu. ) was hybridisable with 200 μg total RNA from tissues and fibroblasts (except for the lungs and heart, where used 10 µg). After digestion by RNase reaction mixture was analyzed by electrophoresis in 4% polyacrylamide/urea gel. Received protected fragments from 575 n. and 449 n. for KL-1 and KL-2, respectively. Autoradiographically exposure continued for 48 or 72 hours, excluding RNA fibroblasts T, where the exposure time was 6 hours.

Fig.21. Panels A-C. the Characteristics of the biosynthesis of KL-1 and KL-2-protein products in COS cells. COS-1 cells were transfusional 5 micrograms KL-1 and KL-2-expressing plasmids using DEAE-dextran. After 72 hours cells were labeled35S-metal with LTOs electrophoresis in SDS page (12%). Egnazia of molecular weight markers is shown in kilodaltons (KD).

Fig. 22. Panels A-C. PMA-induced splitting of KL-1 and KL-2-protein products. COS-1 cells were transfusional 5 micrograms KL-1 and KL-2-expressing plasmids, and after 72 hours, cells were labeled 35S-Met for 30 minutes and then Wednesday for tagging was replaced by the environment: (a) not containing serum; (b) contain complex Herbology ester PMA (1 μm); (C) calcium-ionophor A (1 μm). Supernatant and cell lysates were subjected to thus with rabbit serum against KL. Immunoprecipitate analyzed using LTO-electrophoresis in SDS page (12%). The migration of molecular weight markers is shown in kilodaltons (KD).

Fig. 23. Panels a and B. the characteristics of the biosynthesis KL-SIdand KL-S-protein products in COS cells.

Fig. 24. Determination of biological activity in COS-cell supernatant. Supernatant from COS cells transfected with the KL-1 KL-2, KL-SId- and KL-S-expressing plasmids were analyzed for activity analysis on the proliferation of fat cells. Serial cultivation of supernatants incubated with VMS, and after 24-30 hours of cultivation was determined by the inclusion of3H-thymidine.

Fig rhG-CSF, and rm IL-3 (all at 100 units/ml). Bone marrow is obtained after 4 days from mice treated with 5-FU, were cultured in 60-mm Petri dishes with 2 ml of 0.5% agarose lower layer containing cytokines and covered with a top layer of 1 ml of 0,36% agarose containing 2,5104bone marrow cells. After 12 days of incubation under conditions of low oxygen content of the culture was evaluated on the colony, the diameter of which exceeded more than 0.5 mm

Fig. 26. Analysis of secondary CFU-GM or Delta-cultures, illustrating the degree of increase in GM-CSF-susceptible CFU-GM in the 7-day suspension culture of bone marrow obtained 24 hours after treatment of mice with 5-FU. Cell bone marrow (2/5105ml) were cultured for 7 days with the indicated combinations of cytokine and selected cells again cloned in the analysis of GM-CSF-stimulated colony. The specified zoom ratio defined as the ratio of the number of CFU-GM, allocated in the secondary count of clonogenic analysis to the initial number of CFU-GM, defined in the initial count of clonogenic analysis with GM-CSF; however, rmKL used in the amount of 20 ng/ml, rhII - 6 at 50 ng/ml, rmIL1 in the amount of 100 units/ml, a rhM-CSF or rmlL-3 in quantities of 1000 units/ml

Fig.27. Stimulation of hemopoiesis in bone marrow cultures is L-1 + IL-3 + KL. Cells, 104(after subtraction of granulocytes, lymphocytes) and containing 2.5% NRR-CFU susceptible to IL-1 + IL-3 + KL in the primary count of clonogenic analysis, incubated in suspension; after 7 days in the secondary count of clonogenic analysis determined the total number of cells and HPP-CFU sensitive to IL-1 + IL-3 + KL, or CFU-GM, sensitive to rm GM-CSF. The results were calculated as the ratio of the "output" cells to "input" HPP-CFU.

Fig. 28. The impact of IL-6, IL-1 and KL both separately and in combination on the growth of colonies from normal mouse bone marrow. Control cultures were grown in the absence of any growth factors. Was used seven or combinations of IL-6, IL-1 and KL were tested alone or in combination with CSF, G-CSF, M-CSF, GN-CSF and IL-3. Data were expressed as average values of the average square deviation for the triple duplicates.

Fig. 29. The synergistic action of IL-6, IL-1 and CSF stimulation HPP-CFC, taking place from 5-FU-desensitized bone marrow. Bone marrow was taken at 1-7 days after injections of 5-FU (top to bottom) and were cultured in the presence of G-CSF, M-CSF, and IL-3 + IL-6, IL-1 or IL-6 + IL-1. The data were represented as the total number of CFU-C (HPP-CFC + L-CFC) 1105- 1104(d15) FU (d7) 5-FU bone marrow cells. The results represented the mean values cf. square otklonenijami. As shown in Fig. 28, 40 combinations of cytokines were analyzed for their ability to stimulate CFU-C (HPP-CFC + LPP-CFC), originating from the VM, taken after 5-FU injection. The number of colonies represented the average value of the average square deviation calculated for the triple duplicate cultures 1105(d1) 5-FU BM or 1104(d7) 5-FU BM cells.

Fig.31. The increase in the total number of cells in the cultures requires stimulation combinations of several growth factors. As described in materials and methods, to determine the number of non-stick cells present in the cultures after 7 days of cultivation. The dotted line denotes 1,5105(d1) 5-FU-VMS-cells used for inoculation of cultures. The morphology of the selected cells is discussed in the text. Data represent average values of the average square deviation 2-16 experiments.

Fig. 32. IL-6, IL-2 and KL both separately and in combination have sinergicheskoe action together with SF during the reproduction of LPP-CFC in crops. As described in materials and methods, to calculate the data for L PP-CFC, cultured in the presence of G-CSF, M-SF, GM-CSF, IL-3 or IL-1 + IL-3. -Values were calculated as the average of three duplicate samples of primary and secondary colonies. The results are presented as average values of the ins for LPP-CFC are given in logarithmic scale.

Fig. 33. IL-6, IL-1 and KL, taken separately or in combination, together with F stimulate breeding HPP-CFC in crops. All HPP-CFC were cultured in the presence of II-I + IL-37. -Value was calculated as the average of triple duplicate samples of primary and secondary colonies. The results are presented as average values of the average square deviation 2-11 experiments. It should be noted that the values for HPP-CFC are given in logarithmic scale.

Fig. 34. Precursor cells sensitive to IL-1 + KL do not breed in crops. IL-1 + IL-3 was compared with IL-1 + KL in relation to their effectiveness in stimulating primary and secondary HPP-CFC and LPP-CFC in the analysis. -Value was calculated based on the average values obtained for the three duplicates in the CF-analysis. These data represent the results of one experiment. It should be noted that the values given are in logarithmic scale.

Fig. 35. The number of CFU-S was increased in the cultures. Comparing the values characterizing the increase of HPP-CFC, LPP-CFC and CFU-S, which took place in in vitro analysis or in vivo analysis after administration of 5-FU. -values for in vivo increase of progenitor cells was measured as the ratio of the number of precursor cells was observed 8 days after injection of 5-FU, to the number of cells observed h is sperimental.

Detailed description of the invention

In the present work, a correlation was found between KL and c-kit receptor and on the basis of experiments on the binding and cross-linking has been shown that KL is the ligand of c-kit. To obtain KL-specific cDNA clones used N-terminal protein sequence KL. These cDNA clones were used for studying the relationship of the KL gene with the SI locus, which results demonstrated that KL is encoded by the SI locus.

Recently from the environment, air-conditioned fibroblast Balb/c-T, was isolated hematopoietic factor KL, having biological properties, which, as expected, has a c-kit-ligand [37]. Cleaning KL carried out on the basis of its ability to stimulate the proliferation VMS from normal mice, but not from W-mutant mice in the absence of IL-3. Purified factor stimulates the proliferation of VMS and STS in the absence of IL-3, and this suggests that he plays seems important role in the maturation of fat cells. As for the predicted functions of c-kit in erythropoiesis, it was shown that KL stimulates the area of differentiation of early progenitor cells erythroid series (7-14-day BFU-E) in conjunction with retroporno mass 3 KD and a PI of 3.8; but it is not disulfide-linked dimer, although the characteristics of KL after gel filtration indicated the formation ecovalence linked dimers under physiological conditions.

The predicted amino acid sequence of KL, is derived from nukleinovokisly cDNA sequence suggests that KL is synthesized as a transmembrane protein, and not as a secretory protein. The soluble form of KL can then be obtained by proteolytic cleavage of the membrane-associated form KL. Ligand SF-1-receptor, which is the closest relative of C-kit, has in common with KL topological characteristics, and, as has been shown, it can be proteoliticeski split with the formation of soluble growth factor [44, 45]. Recent analysis of structural features of the KI point to the relationship between KL and CSF-1 (as evidenced by amino acid homology, secondary structure and localization of exons) and illustrated the evolutionary relationship of these two factors, thus confirming the earlier hypothesis about the evolutionary relationships of these two receptor systems.

Alternative were described splanirowannya KL-mRNA, which codeactivity in COS-cells, transfected KL cDNA, and analyzed in several ways Flanagan and others As mentioned above, KL, was synthesized as a transmembrane protein that proteoliticeski was split with the formation of the soluble form of KL. It was shown that the protein product of alternative splanirovano transcript KL, KL-2, which lacks exon encoding the presumed site of proteolytic cleavage, detects as "inverted" characteristics compared to the characteristics of KL-1. In addition, proteolytic cleavage as KL-1 and KL-2 may be regulated by such agents as PMA and potassium-ionophor A. The relative content of KL-1 and KL-2 was determined in various murine tissues of a wide range. The results showed that the expression of KL-1 and KL-2 is under tissue-specific control.

In addition, the identified gene products SId[15]. SIdis a consequence of deletions in KL-sequence that includes the sequence encoding the transmembrane and zitoplazmaticeski domains of the protein, which leads to the formation of biologically active secreted mutant KL-protein. In light of the obtained results were discussed relative role of restwe the invention relates to a purified protein mammals, the corresponding ligand for c-kit. which includes glycosilated of two polypeptides, each of which has a molecular weight of about 30 KD and isoelectric point of about 3.8. Used in the present description, the term "c-kit-ligand" means polypetide or protein, which was also identified as a factor stem cell factor, mast cells, and steel factor. The present invention includes both natural and recombinant forms of the polypeptide and protein ligand, c-kit, and recombinant forms are forms of the protein or polypeptide that is not found in nature, but which is quite identical to the natural forms of c-kit and having similar biological activity. Examples of such polypeptides are polypeptides, designated KL-1.4 and S-KL, however, these examples are not limited to these polypeptides. Examples of the proteins and polypeptides can also serve and derivatives and analogues. In one of the embodiments of the present invention purified protein of a mammal is a mouse protein. In another embodiment, the present invention purified protein mammal is a human protein.

In this application also considers the cell protein. However, the present invention also relates to deglycosylated forms of the protein. In addition, the present invention includes a purified mammalian proteins, glycosylation which is sufficiently similar to the level of glycosylation of natural purified protein of a mammal, the corresponding c-kit-ligand. This protein can be produced by introducing a cysteine cross-linking between the two homodimeric the polypeptides described above, using standartniy techniques, well known to experts.

The present invention also relates to a pharmaceutical composition that contains an effective amount of purified protein of a mammal, the corresponding c-kit-ligand and described above, and a pharmaceutically acceptable carrier.

In addition, the present invention relates to pharmaceutical compositions for the treatment of radiation in mammals, which contains a certain number of hematopoietic factor, effective for the treatment of radiation in mammals; and specified hematopoietic factor selected from G-CSF, GM-CSF and IL-3.

The present invention also relates to pharmaceutical compositions for the authorized above, and an effective amount of EPO (erythropoietin) or IL-3. Examples anemia may serve as, but are not limited to, diamond-blackfan anemia and gipoplasticheskaya anemia. However, this pharmaceutical composition comprising an effective amount of the above composition and an effective amount of G-CSF and GM-CSF, preferably used for the treatment of anemia Blackfan and gipoplasticheskaya anemia. In addition, the present invention relates to a method of treating anemia in a mammal by introducing this mammal the composition described above. The present invention also discloses a pharmaceutical composition effective to increase the survival rate of the bone marrow in mammals during transplantation and containing an effective amount of the pharmaceutical composition described above, and an effective amount of IL-1 or IL-6, is required to increase survival rate of bone marrow from a mammal during transplantation. The authors of the present invention is also the pharmaceutical composition to accelerate the recovery of the bone marrow in mammals in the treatment of radiation, chemical or chemotherapeutical destruction of bone marrow aplasia or myelosuppression, stergade the invention also relates to pharmaceutical compositions for the treatment of acquired immunodeficiency syndrome (AIDS) patients, which contains an effective amount of the pharmaceutical composition described above, and an effective amount of AZT or G-CSF for treatment of AIDS.

In the present invention discloses a composition for treating nervous disorders, which contains an effective amount of the above pharmaceutical compositions for treatment of nervous disorders in mammals.

In addition, the present invention relates to compositions for the treatment of children with symptoms of malformation of the lung, which contains a therapeutically effective amount of a purified protein of a mammal and a pharmaceutically acceptable carrier.

The present invention also relates to a composition for preventing hair loss in an individual, which comprises prophylactically effective amount of a purified protein of a mammal, the corresponding c-kit-ligand, and a pharmaceutically acceptable carrier. In addition, the present invention relates to pharmaceutical compositions for inhibiting the discoloration of the hair of the individual, which contains effective for such inhibiting amount of a purified protein of a mammal, the corresponding c-kit-ligand, and f is ukazannyh disorders by introducing effective composition in the amount necessary for the treatment of this disease.

Used in the present description, the term "individual" means, but is not limited to, a mammal, an animal, a human, mouse or rat. "Mammal" means, but is not limited to, mouse or man.

The present invention relates to a selected nucleic acid molecule that encodes the amino acid sequence corresponding to the c-kit-ligand (KL). Examples of these nucleic acids may serve as, but are not limited to, nucleic acids, denoted KL 1,4, KL-1, KL-2 or S-K1. The present invention also includes nucleic acids that differ from the nucleic acid encoding the above amino acid sequence, but which produce the same phenotypic effect. These changes, but phenotypically equivalent nukleinovokisly molecules hereinafter will be referred to as "equivalent nucleic acids". In addition, the present invention also relates to nukleinovokisly molecules that differ from the above nukleinovokisly molecules fact that they have changes in non-coding regions, but the phenotype of the polypeptide produced by the s hybridized with nukleinovokisly molecule of the present invention, described above. Used in the present description, the term "nucleic acid" means RNA and single-stranded and double-stranded DNA and cDNA. In addition, used in the present description, the term "polypeptide" means any allelic variant, occurring in nature as well as its synthetic recombinant forms.

For the purposes of this invention, the c-kit-ligand (KL) use c-kit-ligand (KI) of human and murine c-kit-ligand (KL).

The present invention also relates to a vector comprising nukleinovokisly molecule that encodes the amino acid sequence corresponding to the c-kit-ligand (KL). Such a vector may be plasmid, viral, Comedy or any other vector.

The present invention also relates to the selected nukleinovokisly molecule, as defined above, which is properly connected to a promoter of RNA transcription, as well as other regulatory sequences. Used in the present description, the term "properly connected" means situated so that initiation of transcription of RNA was carried out under the control of this promoter. Examples of such promoters are the United with the specified promoter, well known in the art. Preferably, these vectors were capable of RNA transcription in vitro. Examples of such vectors are G-series (Promega Biotec, Madison Wl).

In addition, the present invention relates to a system host-vector intended for production of the polypeptide c-kit-ligand (KL) and containing one of the above vectors in an appropriate host. With suitable hosts, which can be used for the purposes of the present invention, include, but are not limited to): mammalian cells or insect cells for expression of the baculovirus. Suitable hosts can be either bacterial cells such as E. cli, or yeast cells.

To highlight protein expressed in E. coli cells E. Li transferout nucleic acid of the present invention that encodes a protein ligand of c-kit. Then, E. coli cells are cultivated in one liter of culture in two different environments, LB or TV, and then centrifuged. Bacterial debris homogenized by a double pass through the cell Frenz under pressure of 200,000 pounds per square inch in 10 ml of lyse buffer (see below). After centrifugation at high speed (120 rpm./min min), supernatant transferred in Deut what W 6 M of guanidine-model HC1 or 8 M urea, followed by dialysis or dilution.

Buffer for lysis: 50 mm Hepes, pH 8.0; 20% glycerol, 150 mm NaCl; 1 mm MDO4; 2 mm DTT; 5 cC EGTA; 20 µg/ml Gnkazy 1.

In the present application is also considered purified soluble polypeptide c-kit-ligand (KL) and the fragment purified soluble polypeptide c-kit-ligand (KL).

In one of the embodiments the present invention relates to a polypeptide c-kit-ligand, corresponding to amino acids 1-164. In another embodiment, the present invention relates to a polypeptide ligand of c-kit, the corresponding amino acids from 1 and up to about 148, or to hybrid polypeptides corresponding to the amino acids from 1 and up to about 148, subsidized by amino acids about 165 to 202 or 205, as well as to hybrid polypeptide corresponding to amino acids 1 and up to about 164, subsidized with amino acids from 177 to approximately 202 or 205.

In the following embodiment of the present invention the polypeptide c-kit-ligand can be a polypeptide corresponding to amino acids 1 to about 164 connected to a biologically active binding site. Examples of such biologically active binding sites include, but are not limited to, amino acids, corresponding to the binding site for ssit cell activation. See, for example, the U.S. patents 4578079, 4614517 and 4792525, issued March 25, 1986, September 30, 1986, and December 20, 1988, respectively.

In one of the embodiments of the present invention are soluble polypeptide c-kit-ligand (KL) is subjected to conjugation with visualizing agent. Imaging agents are well known in the art, and examples of such agents can serve as radioactive isotopes, dyes, or enzymes such as peroxidase or alkaline phosphatase. Suitable radioisotopes are125I32P,35S and other

These conjugated polypeptides can be used to detect cells in vitro or in vivo), which Express c-kit receptor protein. When detecting in vitro a sample of the tested cells or tissues are subjected to contact with the conjugated polypeptide under appropriate conditions conducive to the binding of this conjugated polypeptide with c-kit-receptor present on the cell surface or tissue, after which unbound conjugated polypeptide remove and record the presence of bound anywayawesome polypeptide, thereby detecting cells and tissues that Express c-kit receptor protein.

Alternatively, konbit introduced a sufficient amount of conjugated polypeptide, which may vary within a wide range depending on size, mass and other characteristics of the patient. However, this number can easily be determined by any expert.

After the introduction of the conjugated polypeptide that is associated with any c-kit-receptor present on the surface of cells or tissue, detect by intracellular visualization.

In the method of the present invention intracellular visualization can be carried out by any of the many existing visualization methods, for example by detecting and imaging radiation of the radioactive isotope. For example, if a radioactive isotope is iodine, namely 125I, the detection and visualization of the radiation of this isotope can be carried out using a gamma camera to detect the emission of radioactive iodine.

In addition, the soluble fragment of the polypeptide c-kit can be anywhereman with a therapeutic agent, such as toxins, chemotherapeutic agents or radioactive isotopes. Thus, the conjugated molecule, administered to the patient in an effective amount, acts as a tissue-specific delivery system for therapeutic agent is odwzorowania polypeptide c-kit-ligand (KL), providing the cultivation system "vector-host" described above, under appropriate conditions, ensuring the production and secretion of the polypeptide c-kit-ligand (KL).

In addition, the present invention relates to a polypeptide c-kit-ligand (KL) produced by the above method.

The present invention also refers to antagonists of c-kit-ligand. These antagonists can be small molecules, detected by the test-screening for the presence of c-kit-receptor. Alternatively, they can be antimyeloma nucleic acids, DNA, RNA, based on the ribose or other Zaharenko frame with thiophosphate, methylphosphate, methylphosphonate bonds between the sugars. Antisense molecules block the broadcast of c-kit-ligand in vivo.

In the present invention also includes a soluble mutant antagonist of c-kit-ligand (KL), and this mutant polypeptide retains its ability to bind c-kit receptor, but blocks the biological response, which is mediated by the binding of a functional ligand to the receptor. Thus, these mutated polypeptide c-kit-ligand (KL) act as antagonists against biologica ligands with c-kit receptor. KL-antagonists can be obtained by non-specific mutagenesis. As an effective antagonist can be used mutated or deficitobama KL molecule that does not have the ability to dimerization. KL reveal a high degree of homology with M-CSF containing multiple spirals, which, obviously, play an important role for dimerization [102]. Site-directed mutagenesis performed in these helical regions, it is possible to block the ability to dimerization. Alternatively, the mutated KL can form heterodimer with normal functioning KL, but this heterodimer not the way to activate c-kit receptor. Because c-kit-receptor itself requires dimerization to form the active kinase, an effective antagonist may be soluble mutated KL, which by binding to c-kit receptor, is already blocking the dimerization of the receptor.

The present invention also relates to pharmaceutical compositions containing the c-kit-ligand (KL), purified by the authors of the present invention or produced recombinant methods, and a pharmaceutically acceptable carrier. The specified c-kit-ligand can be selected soluble c-kit-ligand present from this

the description of the term "pharmaceutically acceptable carrier" means any of the standard pharmaceutical carrier, such as phosphate buffer solution, water, and emulsions, such as emulsion oil/water or water/oil, and various types of wetting agents. In these pharmaceutical carriers can also be included aerosollaboratoriet substances.

KL-antagonists, described above, can be used to treat a number of diseases, including diseases such as asthma, allergies, anaphylaxis, allergic asthma, arthritis, for example rheumatoid arthritis, papillary conjunctivitis, leukemia, melanoma, skin allergic reactions, scleroderma.

The present invention also refers to a substance capable of specific complex formation with protein c-kit-ligand (KL) or its fragment, described above. The present invention also refers to a substance capable of specific formation of the complex with the receptor protein c-kit-ligand (KL). In one of the embodiments of the present invention the specified substance is a monoclonal antibody, such as monoclonal antibody man.

In addition, the present invention relates is hearing the interaction of the sample cell, functions which must be modified with an effective amount of the above pharmaceutical compositions necessary to modify the biological function of cells. Examples of biological functions that can be modified include, but are not limited to, functions such as intercellular communication, reproduction of cells expressing c-kit, and in vitro fertilization. This method can be performed in vitro or in vivo. When in vivo implementation of this method, the patient is administered an effective amount of the above pharmaceutical compositions necessary to modify the biological function associated with c-kit function.

Another variant of the present invention relates to ex vivo-methods and compositions containing KL in the media, suitable for ex vivo use.

Those methods are:

1. a way to enhance transfection of early hematopoietic precursor cells via gene, namely, that first early gomeopaticheskii cells subjected to contact with a composition containing KL and hematopoietic factor, and then the cells cultured in stage (a), transferout genome;

2. the method of gene transfer MLEKO the Oia with composition containing KL; (b) cells from (a) transferout genome; (C) cells, transfetsirovannyh in (b), is administered to the mammal. In the described methods as gene can be used antisense DNA or RNA.

Composition containing KL, can be used to increase the ex-vivo levels of peripheral blood and an effective amount of hematopoietic factor (or factors) growth. For these purposes, especially preferred are hematopoietic growth factor, IL-1, IL-3, IL-6, G-CSF, GM-CSF, or combinations thereof (see Fig. 26). In the present application is also considered a way to increase the level of peripheral blood.

In addition, this application discusses how and KL-containing compositions for increasing levels of platelets or other cells types (IL-6 are particularly preferred).

The present invention also relates to a method of modifying the biological functions associated with c-kit-cell activity by interaction of cells with KL. Such a cell can be a cell expressing c-kit, or a hematopoietic cell, or the cell that is involved in in vitro-fertilization.

The present invention also relates to a method of stimulating proliferation of fat citationstyle, effective for proliferation of fat cells in the patient. Methods of introduction of the compositions is well known to every expert, such as orally, intravenously or parenterally, and other methods of introduction. The dose of injected composition should be such that it stimulated poliferation fat cells. This introduction can be carried out continuously or intermittently, so that the amount of composition introduced to the patient effective to stimulate proliferation of fat cells.

In addition, the present invention relates to a method of inducing differentiation of fat cells or precursor cells erythroid series in a patient, which consists in the fact that the patient is given the above pharmaceutical composition in an amount effective to induce differentiation of fat cells or erythroid progenitor cells. Methods of introduction of the compositions is well known to every expert, and examples of such methods include oral, intravenous, or parenteral administration. The dose of injected composition should be such that it contributed to the differentiation of fat cells or erythroid progenitor cells. While the introduction of m is effective to induce differentiation of fat cells or erythroid progenitor cells.

The present invention also relates to a method of stimulating or increasing levels of progenitor cells using c-kit-ligand, alone or in combination. Particularly effective are combinations of c-kit-ligand, G-CSF, GM-CSF, IL-1, IL-3, IL-6, IL-7 and MIPI. The most effective is a combination of KL + IL-1, IL-3 and IL-6. However, IL-1, IL-3, IL-6 and GM-CSF, taken separately, have moderate efficiency. This, in particular, is illustrated in the analysis on the growth of colony-forming cells with high proliferative potential, which was conducted with the use of bone treated with 5-fluorouracil These combinations can be used in vivo, in vitro, and ex vivo.

In addition, the present invention relates to a method of facilitating bone marrow transplantation or treatment of leukemia patients, namely, that this patient is given the above pharmaceutical composition in an amount effective to facilitate bone marrow transplantation or for the treatment of leukemia. Methods of introduction of the compositions is well known to every expert, and examples of such methods is oral, intravenous, or parenteral administration. The dose of injected composition should be such that it contributed to the discontinuously or periodically so the quantity of the composition introduced to the patient to be effective. This method is particularly effective for the treatment of chronic myeloid leukemia, c-kit-ligand contributes to the increase in the rate of growth of white blood cells, thereby increasing their susceptibility to chimiotherapie.

The present invention also relates to a method of treating melanoma in a patient, which consists in the fact that this patient is administered an effective amount of the above pharmaceutical compositions for treatment of melanoma. This composition may be introduced by standard methods, such as oral, intravenous, parenteral, or other method of administration. The dose of injected composition should be sufficiently effective for the treatment of melanoma. While the introduction of the composition can be carried out continuously or intermittently, so that the number entered to the patient the composition was effective.

Soluble polypeptide c-kit-ligand can also be mutated so as to block the biological activity of c-kit, while maintaining its ability to bind with c-kit. Thus, the present invention relates to a method of treating Allergy in a patient, which consists in the fact that this patient is acceptable carrier. This composition can be made into aerosol form, which is introduced by spraying water forms mutated antagonist c-kit-ligand. K1-antagonist, described above, is also effective against allergies, and also in aerosol form.

The pharmaceutical composition for external use containing antagonist of c-kit-ligand, is an effective tool to use against arthritis, rheumatoid arthritis, scleroderma, acute dermal allergic reactions. Specified antagonist of c-kit-ligand can also be effective against allergic conjunctivitis, postalservices to tissue damage, or it can be used as a prophylactic against anaphylactic shock. Because mast cells mediate the histamine response, the antagonist of c-kit or antisense molecule complementary to c-kit-ligand, should be effective to block histamine-mediated responses, including Allergy and the secretion of gastric juice.

Antagonist of c-kit should be effective for the treatment of melanoma, because the melanocytes in their development largely depend on KL. Similarly, KL-antagonists can be used for l is effective for the treatment of allergies, may vary for each individual patient and for a specific type of Allergy. While the introduction of the composition can be carried out continuously or intermittently, so that the amount of composition introduced to the patient to be effective.

In addition, the present invention relates to a method of determining the biological activity of the polypeptide c-kit-ligand (KL), namely, that normal fat cells in the bone marrow incubated with the sample polypeptide c-kit-ligand (KL) under conditions suitable for inducing the proliferation of normal fat cells of the bone marrow; double-mutated fat bone marrow cells incubated with the sample polypeptide c-kit-ligand (KL) in suitable conditions; each of the products obtained incubated with3H-thymidine; determine the amount of thymidine incorporated into the DNA of normal fat cells of the bone marrow and compare the amount of thymidine incorporated into the normal fat cells in the bone marrow, with the amount of thymidine incorporated in the double-mutated fat cells in the bone marrow, resulting assess the biological activity of the polypeptide c-kit-ligand (KL).

In the description of the present application with reference to specific nucleate specific nucleotides were used following standard notation: cytosine; And adenosine; T is thymidine; G - guanosine; U-uracil.

Experiment 1. Purification of c-kit-ligand

Experimental materials

Mouse embryos

Mouse W BB6 +/+ and W/Wv. C57B16W/+ and WB W/+ were obtained from the Jackson laboratory (Jackson Labkratory, Bar Harbor ME). Heterozygous W41/+ mice were kindly provided by Or. J. Barker (Jackson Laboratory) and were maintained in a colony obtained by the applicants by mating of siblings. Of the embryos on day 14-15 pregnancy resulting from crossing W/+ animals were removed liver. W/W-embryos were identified by their pale color and small size of the liver compared with other embryos (W/+ and +/+) litter. Identification of these embryos was confirmed by analysis of c-kit protein in mast cells derived from each embryo [38].

Culture of fat cells, receiving peritoneal mast cells and flow cytometry

Fat cells originating from the bone marrow of adult mice, and liver cells of the embryo at 14-15 days of gestation were cultured in medium RPMI-1640, supplemented with 10% fetal calf serum (FCS); the environment, air-conditioned EN-3V-cells; amino acids, non-essential; sodium pyruvate and 2-mercapto-ethanol (RPMI-medium is etoc less than 7105cells/ml of the Compound of the fat cells in the cultures was determined weekly by colouring cytopenia drugs 1% toluidine blue in methanol. After 4 hours of culture usually contained more than 95% of the fat cells, and these cultures were used for analysis of proliferation. Peritoneally fat cells were obtained from 57L/b-mice by lavage of peritoneal cavity 7-10 milliliters of RPMI-C. Mast cells were purified by centrifugation in a density gradient of 22% Metrizamide (Nycomed, Oslo, Norway) in PBS not containing CA++and Mg++basically, as described previously [61] . Mast cells were stained with 1% toluidine blue in methanol for 5 minutes, within 5 minutes, washed in water, and berberine sulfate using standard procedures [62]. Fat cells were subjected to staining with c-kit-specific rabbit anticigarette that recognizes the extracellular determinate C-kit, and analyzed on a FACSCAN (Becton Dickinson) [38].

Analysis on the proliferation of fat cells

Fat cells three times washed in RPMI to remove IL-3, and were cultured at a concentration of 510 cells/ml in RPMI-C in a volume of 0.2 ml in 96-well tablets with twofold serial dilutions of the test samples. The plates were incubated at 37oWith entrances were collected on glass fiber filters, then determined the level of incorporation of thymidine into DNA.

Receiving environment, conditioned by fibroblasts

Balb/T-cells [1] were cultured to the condition of continuity of the modified according to the method of Dulbecco environment the Needle (D-MEM), supplemented with 10% calf serum (CS), penicillin and streptomycin, in a rotating vials (roller bottles). Then the medium was removed, and the cells twice washed formato-buffer solution (PBS). Then add MEM, not containing CS, and after three days was collected, air-conditioned environment. After that, the cells were fed with medium containing serum, in 1-2 days, then washed with serum-free medium, again fueled, but serum-free medium, and after 3 days were collected in the second game, air-conditioned environment. Conditioned medium (CM) was centrifuged at 2500 rpm./min for 15 minutes to remove cells, and then filtered through 0.45 μm filter and frozen at 4oC. thereafter, the conditioned medium was concentrated to 100-200-fold reduction with use of devices Pellicon ultrafiltration, and then the device for mixing Amicon, when this membrane had a cutoff of 10,000 kDa.

Column chromatography

Will chromatogra PBS. 50-80 ml FCM-concentrate was loaded into the column after equilibration for 1 hour stream containing the active material, was collected and concentrated to a volume of 15-20 ml tube for dialysis with PEG 8000.

Gel chromatography was performed on a column (2,690 cm) AS Ultrogel (LKB, Rockiand, MD), which was balanced by PBS and grooved with taps molecular weight, bovine serum albumin (Mr 68000), chymotrypsin (Mr 25700) and ribonuclease A (Mr 14300) (all of these proteins were obtained from Pharmacia, Piscataway, NJ). The concentrate from the column with a blue agarose was loaded into a column for gel filtration, the flow rate was brought up to 37.5 ml/h, and 7.5 ml fractions were collected.

Anion-exchange and reversed-phase high-performance liquid chromatography (URGH)

High-performance liquid chromatography was performed using Waters HPLC (regulator W 600E Powerline, multi-wave detector with software control E, automated workstation 810, Baselime, Bedford MA). Active fractions from gel filtration were dialyzed in 0.05 M Tris-HCl pH 7.8 and loaded into URGH-column Protein-Pak DEAE-5PW (7.5 to mm,5, see Waters), balanced 0.05 M Tris-HCl, pH of 7.8. Associated proteins were suirable linear gradient (0-->0.05 M) Tris-HCl, pH of 7.8. Associated proteins were suirable line is>/P>Reversed-phase URGH was performed using prepreparation and analytical C18-column from Vydac. Columns used two buffers: buffer A-100 mm ammonium acetate (pH 6.0), and the buffer In - 1-propanol. Biologically active fractions from the anion exchange column were combined and loaded into prepreparation18a column. Associated proteins were suirable steep gradient (0 - 23%) 1-propanol within the first 10 minutes, and 23-33% gradient of 1-propanol for 70 minutes. The flow rate is brought up to 2 ml/min and 2 ml fractions were collected. Biologically active fractions were pooled and diluted (1:1) buffer a And then loaded into an analytical reversed-phase C18-column. proteins were suirable steep gradient (0-26%) 1-propanol for 10 minutes, and then for 70 minutes was suirable gentle gradient (26-33%) 1-propanol. When the flow rate was 1 ml/min Fraction of 1 ml were collected, separation on an analytical reversed-phase C4 column was performed using a linear gradient of acetonitrile (0-80%) in water with 0.1%TFA.

Isoelectric focusing

To 1 ml of partially purified KL was added 20% glycerol (by volume) and 2% ampain (by volume) at pH 3.5-10 (LKB, Gaithersburg, MD), Glycerol gradient plottest gradient and subjected to isoelectric focusing (IEF) (2000, 24 h, 4oC). Then 5 ml fractions were collected and each fraction was determined by pH. These fractions were dialyzed against RPMI-C, and then analyzed for biological activity.

Analysis of erythroid precursors

Cells from Mature bone marrow, spleen and liver of the 14-day embryos were seeded at a density of 105, 106and 107cells/ml, respectively on Wednesday, Dulbecco, modified by the method of Claims and containing 1.2% methylcellulose, 30% FCS, 100 μm 2-mercaptoethanol, recombinant human erythropoietin (2 units/ml, Amgen, Thousand Oaks, CA) (Iscove, 1978; Nocka and Pelus, 1987). Cultures were incubated for 7 days at 37oAnd hemoglobinuria in bordoloni analyzed for investitsionno microscope. To optimize growth conditions for cultures of bone marrow cells was added to 0.1 mm hemin (Cooec). Purified KL, IL-3 (either in the form of WEHI-3 CM (10%, vol./about.) or recombinant murine IL-3 (50 µg/ml, Genzyme, Cambride) was added, as indicated.

Experimental methods

A short analysis on the proliferation of fat cells to detect fibroblast growth factor

For identifying and measuring the activity broblast growth factor, which promotes the proliferation of normal (not W/Wwho entirelynew Wednesday, air-conditioned fibroblasts (FCM) or WEHI-CM (IL-3) and after 24 hours of incubation was determined by the level of inclusion of H-thymidine. Answers VMS obtained from normal (+/+) and mutant mice, IL-3 were identical (Fig. 1). In contrast, 20-fold concentrated medium conditioned by fibroblasts, contributed to proliferatio + / + mast cells, but cells were observed slight proliferation. Concentrated FCM researchers for its ability to stimulate the proliferation of other IL-3-dependent cells. As you know, myeloid cells 321 do not contain c-kit gene products [35] . Environment FCM cell proliferation 321 was not observed, and WEHI-3 CM was observed in normal proliferation (not shown). Summarizing the results obtained and taking into account the absence of both alleles [38] , we can assume that the FCM-activity depends on the functional expression in fat cells (VMS), and therefore, the FCM may be the ligand for c-kit-receptor. In addition, FCM-activity differs from IL-3. Therefore, normal and W-mutant mast cells are simple and specific analytical system for treatment of the proposed c-kit-ligand (KL) of the environment, air-conditioned fibrilationonline fibroblasts alb/T, concentrated by ultrafiltration (50-fold concentration). The obtained concentrate was passed through a column of blue agarose, balanced PBS, and a stream containing stimulating the fat cells activity were collected and concentrated with polyethylene glycol. In addition to the determination of biological activity using fat cells all peak fractions were also analyzed using W/Wv-fat cells, where there is little activity. The material from the column with a blue agarose was fractionally by gel filtration using a column ACA 54 (Fig. 2A). Biological activity was suirable as major and minor peaks corresponding 55-70 kDa and 30 kDa, respectively. Fractions of the main peak were pooled, realizowany and fractionally using FP1-chromatography (liquid Express chromatography of proteins on a column of DEAE-PW with a gradient of NaCl (Fig. 2B). The activity was suirable when 0,11 M NaCl with FPLC-column. Peak fractions were pooled and subjected to VRGH-chromatography using prepreparation C18-column and a gradient of ammonium acetate /n-propanol (Fig. 2C). Active material, suirvey 30% n-propanol from prepreparation C18-column, diluted with buffer A(1:1) and snowl major peak (O,P. 280 nm) in the elution profile, again suirable 30% n-propanol. Similar results were obtained using a C4 column and N2Oh and acetonitrile containing 0.1% TFA as solvent (Fig. 3B). LTOs electrophoresis in SDS page active fractions obtained by separation using both solvent system, and silver staining detected a single band with a mobility corresponding to a molecular weight of 28 to 30 KD. The presence and intensity of this band is directly correlated with the peak of biological activity (Fig. 3). While no significant differences in the migration of a specified band in the recovery and nevosstanovlenie conditions were not observed, suggesting that KL is not a dimer with by a disulfide bond (3C). Three discrete sample was analyzed using restorative and nevosstanovlenie LTOs electrophoresis in SDS page, in the result, it was found that the purified material is heterogeneous in size. The full amount of protein, as determined by optical density at 280 nm corresponds to the amount of protein determined by silver staining relative to BSA used as a standard. As shown in Tabinay full activity was 47%. In the analysis conducted by the applicant in the amount of 0.2 ml, the half maximal proliferation + / + mast cells was defined as 50% activity corresponded to 0.5 ng of protein. Isoelectric focusing of partially purified material (after ion-exchange chromatography) revealed a major peak of activity in the pH range of 3.7-3.9, which indicates that the isoelectric point for KL is of 3.7-3.9.

Proliferatively response to KL fat cells with different C-kit/ W-mutations

Purified KL tested for their ability to stimulate the proliferation of fat cells obtained from wild-type animals, as well as from homozygous and heterozygous for alleles W, Wvand W41. The original W-allele defines non-functional c-kit receptor, and animals homozygous for the W allele die in utero or have severe anemia; and fat cells, derived from W/W-embryos are capable of proliferation by co-cultivation with alb/T-fibroblasts [63, 38]. Both Wvand W41allele define partially defective c-kit receptor, and homozygous mutant animals are viable [64, 65, 38]. Wv-homozygous animals have severe macrocytic anemia, and the fat cells these is th less strong41-allele have moderate form of anomie, and fat cells of these animals show an intermediate response in the analysis with the joint cultivation. Homozygous and heterozygous mutants and + / + mast cells derived from bone marrow W and W41-allelic animals and from the liver of 14-day embryo W-allelic animal, were described previously [38]. W/W fat cells, derived from embryonic liver did not show proliferation in response to KL, whereas heterozygous (W/+) and normal (+/+) mast cells were found similar proliferative response to KL (Fig. 4). Fat cells originating from bone marrow Wv/Wvmice were given very little response to KL, although some degree of proliferation (10% + / + - values) was observed at 100 units/ml (Fig. 4). In contrast, heterozygous W/+ mast cells W3+ mast cells showed an intermediate response (40%), which is consistent with the dominant characteristics of this mutation. W41/W41and W41/ + mast cells also had insufficient capacity for proliferation with KL, albeit less pronounced than fat cells bearing W and Wvalleles, which corresponds to the in vivo phenotype of this mutation (Fig. 4). The results illustrated sawh mutations. In contrast, the proliferative response of mutant fat cells WEHI-3 CM (11-3) are not affected by the different W-mutations.

KL stimulates the proliferation of peritoneal mast cells

Fat cells in the abdominal cavity (PMC) have been thoroughly investigated and in contrast VMS are fat cells, like mast cells of connective tissue [66]. PMC is not capable of proliferation only in response to IL-3, but their Mature phenotype and viability can be maintained by co-culture with NlH/T-fibroblasts [67]. It was therefore interesting to establish whether K1 to stimulate the proliferation of PMC. First, we conducted an experiment to determine whether C-kit expressed in PMC. For this peritoneal mast cells were purified by precipitation in the gradient metrizamide, and then by immunofluorescence assay using serum against C-kit or normal rabbit serum were analyzed for expression of C-kit on the cell surface. Purity PMC-preparations was 90-98%, which was established by staining toluidine blue and sulphate of berberine. Sulphate of berberine paint proteoglycans, such as heparin in granules fat latoyah membranes contain mainly chondroitin sulfate (di-B/E-proteoglycan), instead of heparin proteoglycan [67], and therefore, the sulphate of berberine does not stain the granules in VMS (Fig. 5A). Analysis of the expression of c-kit by flow cytometry showed that virtually all RMS Express C-kit at levels similar to the levels observed in VMS (Fig. 5B). Then conducted a study to determine whether KL survival PMC or KL can stimulate the proliferation of PMC (Fig.5C). Cultivation PMC only in one environment or adding WEHI-3 CM at a concentration that is optimal for VMMS, leads to loss of viability of RMS within 3-4 days, although a few cells survived in WENI-3CM over a longer period. Cultivation of RMS in the presence of KL contributed to the preservation of their viability and in two weeks the number of cells increased from 5000 to 60000. A similar increase in the number VMSS was observed in response to KL. In contrast to RMS that did not show a proliferative response in WEHI-3CM, VMS, as expected, the proliferative response was observed. After a week and two weeks of cultivation, the cells were stained with toluidine blue and sulphate of berberine. Mature phenotype PMC maintained in culture with 100% of the cells increased both dyes, although they will okrashivaemuyu the formation of early precursor cells erythroid series, forming an "explosion-like" colonies (burst-colonies) (BFU-E)

An important feature of the W mutation is their impact on the line of differentiation of erythroid cells. In vivo consequences of this mutation are expressed in macrocytic anemia, which is lethal for homozygotes most strong alleles [47, 65]. Analysis of populations of erythroid progenitor cells in the bone marrow W/Wv-mice showed a slight decrease in BFU-E and CFU-E [68, 69] . In the liver W/W-embryos, the number of BFU-E remained unchanged, however, a significant increase in the number of BFU-E suggests that C-kit plays a role in certain stages of maturation of erythroid cells, obviously, before the CFU-E stage [35]. To assess the role of KL in erythropoiesis and subsequent determination of its relationship with c-kit-receptor investigated the effect of KL on the formation of BFU-e cells in the bone marrow, spleen and liver of the embryo were seeded using standard culturing conditions, in the presence (or absence) of KL, erythropoietin, and WENI-3CM. On the 7th day of cultivation, BFU-E were analyzed. In the absence of erythropoietin, but in the presence of WEHI-3 CM or KL growth of erythroid cells was not observed. In the presence of erythropoietin BFU-E spleen were stimulated KL dozozavisimoe stimulation at 2.5 ng KL/ml (Fig. 6). In addition to the impact on the number of BFU-E were also registered a sharp increase in the average BFU-E, due to KL. The number of BFU-E, produced using spleen cells together with K1+ erythropoietin was similar to the number of BFU-E, obtained using (WEHI-3 CM+erythropoietin). In contrast, KL-erythropoietin did not stimulate the proliferation of BFU-E from bone marrow cells, whereas WEHI+ erythropoietin induced the formation of BFU-E from 103bone marrow cells. It was also evaluated the effect of KL on the liver cells of the 14-day fetus, and the results were similar to the results obtained for cells of the spleen. When using only one of erythropoietin has been a significant number of BFU-E from liver cells of the fetus, but this number was increased from 62 to 20 5 using 2.5 ng/ml KL. In the presence of WEHI-3CM and erythropoietin was observed 183 BFU-E cells of the liver of the fetus.

To further assess the relationship KL with c-kit in relation to the differentiation of erythroid cells studies have been conducted to determine contribute KL education erythroid barstobrick early progenitor cells (BFU-E) from the cells of the embryonic liver W/W mice. W/W and E/+ or + / + cells of the liver were Plantago embryo was 8 times less compared with normal embryos. The number of BFU-E from W/W and E/+ or +/+ - embryonic liver was similar in culture with IL-3 + EPO, and lower under cultivation with only one erythropoietin (Fig. 7). For W/W - fetal liver cells KL did not stimulate BFU-E above those levels, which were observed under cultivation with only one erythropoietin, whereas the number of KL-dependent BFU-E from W/+ or + / + cells of the liver was the same as the number obtained under cultivation with erythropoietin + 1L-3. These results suggest that the sensitivity of erythroid precursors to KL depends on the function of c-kit.

The study of binding to purified KL

Purified KL subjected tagging125I chloraminated method for iodination, resulting in received highly specific activity, i.e., up to 2,8105imp./min/ng.

Using labeled KL, it was found that KL specifically associated with fat cells. But it was not fixed linking W/W fat cells, however, there was a good binding to fat cells from the same litter. After binding to mast cells KL were subjected to co-deposition with anticorodal against c-kit. In addition, linking KL W-mutant

Determination of the peptide sequence of the C-kit-ligand

Protein C-kit-receptor was isolated as described above and the sequence of this protein was determined by standard methods.

Below shows the N-terminal amino acid sequence of the protein (using single letter abbreviations):

KEIXGNPVTDNVKDITKLVANLPNDNYMITLNYVAGMXVLP,

where K= lysine, E=glutamic acid, I=isoleucine, X= unknown amino acid, G=glycine, N=aspargine, P=Proline, V= valine, T=threonine, D=aspartic acid, K=Latin, A=alanine, Y=tyrosine and M=methionine.

Discussion experiments

The discovery of the fact that the W-locus and protooncogene c-kit are allelic, was an important information regarding the functions of c-kit in the process of development of the animal body, as well as in the adult animal. Information about the function of c-kit, in turn, provides an important clue what types of tissues and cells produce the ligand of c-kit-receptor. To identify c-kit ligand growth factor, denoted by KL, was purified from the conditioned medium of fibroblasts alb/T, i.e. cells, "suspects" in the production of c-kit-ligand and having biological properties, which are expected to have a c-kit-ligand in relation to the biology of cells from itapoa the script as opposed to CSG-1, PDCF-B, which possess this property [70,71] (although gel filtration KL in PBS and pointed to the dimensions 55-70 kDa, indicating the presence of ecovalence linked dimers under physiological conditions.) KL is different from other hematopoietic growth factors, their effect on fat cells, such as IL-3 and IL-4 (due to its ability to stimulate the proliferation VMS and purified peritoneal mast cells (STMS), except VMS from W-mutant mice). Sources for hematopoietic growth factors G-CSF, GM-CSF, CSG-1, L1F and IL-6 are fibroblasts alb/T, but none of them have activity KL. In addition, preliminary results obtained in the determination of protein sequence KL, showed that KL is different from known protein sequences.

On the basis of the fact that W-mice lacking mast cells [72,73] , the conclusion was made about the important role of C-kit and its ligand in proliferation, differentiation and/or survival of mast cells in vivo. However, it is not known at what stage (or stages) differentiation of fat cells requires the involvement of C-kit. Mast cells derived in vitro from bone marrow, liver, embryo or spleen IL-3, similar to the fat cells if Inoi stage differeciate [66] . Probably, the presence of c-kit is not required for producing VMS of hematopoietic precursor cells, because IL-3 - dependent mast cells can be generated with the same efficiency from the bone marrow or the liver of the embryo as normal and W-mutant mice [60]. Illustration of the fact that the expression of c-kit in VMS and STMS/PMC and the corresponding sensitivity VMS and Mature STEMS/PMC and KL suggests that c-kit plays a role at different stages of differentiation of fat cells. It was also shown that in addition to fibroblasts combination of IL-3 and IL-4; IL-3 and PMA or cross-linking of IgE receptors can stimulate in vitro proliferation STMS[74, 75, 76, 77, 78]. In contrast to these modulators of biological responses, which are mediators of inflammatory and allergic reactions, KL itself in the presence of FC is capable of stimulating the proliferation STMS. So KL can be capable of proliferation and differentiation of fat cells, which does not depend on these immune responses for its production and impact on the target cells.

Impact W-mutations on erythropoiesis shows the important role of c-kit in the maturation of erythroid cells [80, 68, 69]. The comparative is ologit, in the absence of C-kit function maturation proceeds normally only up to the BFU-E stage, and CGU-E-stage of this development is inhibited [35] . In vitro, this phenomenon can be eliminated by introducing into the culture medium IL-3, which together with erythropoietin stimulates the maturation of BFU-E from W/W and + / + bone marrow [78]. The role of IL-3 in this process in vivo is still unknown, and therefore, c-kit may play a crucial role at this stage of differentiation of erythroid cells. This role of C-kit at this stage of differentiation of erythroid cells is consistent also the ability to KL to stimulate the formation of early barstobrick erythroid precursor cells, derived from cells of the spleen and fetal liver in the presence of erythropoietin. In addition, the ability of KL to stimulate W/W - BFU-E suggests that the function of C-kit is required for KL-mediated formation of BFU-E, and this means that c-kit function is required for KL-mediated proliferation of fat cells. Bartolomaeus action Balb/3T3 cells on the differentiation of BFU-E cells embryonic liver was described previously [79]. In all likelihood, KL is sensitive to burtocthebloody activity Balb/C cells. An interesting aspect of the described study result suggests that that BFU-E in the liver of the embryo, spleen and bone marrow Mature organism differ in terms of their growth. Recently experiments have shown that KL can stimulate the formation of early erythroid-multipotential precursor in the bone marrow, which appears in the culture at a later time (14-20 days). To illustrate directly the effect of KL on the formation of BFU-E and to exclude the possible participation of A-cells or other endogenous factors of growth it was necessary to conduct experiments using purified populations of progenitor cells.

W-mutations in addition to the failure of erythropoiesis and development of fat cells, obviously, affect the compartmentalization of stem cells in the hematopoietic system. Such affected populations can be colony-forming units (cells) spleen (CFU-S), which produce myeloid colonies in the spleen of mice exposed to lethal doses, and long-lived the repopulation required for differentiation of various cells[81, 46, 47, 81, 82]. In addition, it would be interesting to know whether any action is self-sustaining KL or differentiation of populations gomeopaticheskih stem cells can only influence stage) compartmental stem cells, in which (or in which) involved c-kit/W-gene product.

Mutations in the steel locus (SI) of mouse contribute to the production of pleiotropic phenotypes in geomoose, melanogenesis and gametogenesis, which are similar to the phenotypes of mice carrying W mutations [47, 51]. However, unlike W-mutations, SI-mutations affect the cellular microenvironment of the target for mutation, and are not cell-Autonomous [46]. On the basis of parallel and complementary effects of W and SI mutation was assumed that the SI gene encodes the ligand of c-kit-receptor or gene product directly associated with the production and/or function of this ligand [9]. According to this assumption fibroblasts SI/SId-embryo or air-conditioned environment from SI/SId-fibroblasts are not able to support the proliferation VMS and predecessors fat cells respectively and, in all likelihood, will not produce functional KL [16, 84]. If KL is a ligand of C-kit receptor, the molecular analysis will allow us to accurately establish the identity of KL with the gene product of the SI locus and, in addition, to ensure that the introduction of KL mice bearing SI-mutations. would eliminate at least some of the symptoms of this mutation.

Experiment 2. Selection nukleinovokisly sequence

Experimental materials

Mouse and tissue culture

Mouse WBB6+/+, C57BL/7J, C57BL/67 Wv/+, WB6W/+, Sniv/FeJ-a/and GaJ SI Hm, and M. spretus) was obtained from the Jackson Laboratory (The Jackson Laboratory, Bar Harbor, Me). For interspecific crosses were paired females 57L/6J and males of M. spretus. The offspring from this mating were then evaluated on the inheritance of alleles 57L/6J or M. spretus (see below). Females (C57BL/6JM.spretus) F1 offspring were subjected to reverse the crossing with 57BL/6J-males.

The fat cells from the bone marrow adult +/+b Wv/Wvand W/+ mice and liver 15-14 days W/W-embryos were cultured in medium RPMI 1640, supplemented with 10% fetal calf serum (FCS), environment, air-conditioned WEHI-3B cells, amino acids, non-essential, sodium pyruvate and 2-mercaptoethanol (RPMI Complete) [36, 60]. Cells of Balb/c 3T3 [1] received from Paul O'donnell (Sloan-Kettiring Institute new York), and cultured in minimum maintenance medium (MEM), modified by way of Dulbecco and supplemented with 10% calf of Siva KL

KL was isolated from the environment, air-conditioned Balb/c 3T3 cells, with analysis on the proliferation of fat cells, as described in the literature [37]. Then the conditioned medium was concentrated to 100-200-fold decrease volume using the device for Pellicon ultrafiltration and cell mixer Amicon. Thereafter, the concentrate was chromatographically on blue agarose (Bethesda Reseath Laboratories, Gainhersburg, MD) and the stream containing the active material, was concentrated in the tube for dialysis with polyethylene glycol 8000, and then fractionally by gel-chromatography on a column of gel AS Ultrogel (LKB, Rockland, MD). The biologically active material was suirable as major and minor peaks corresponding 55-70 KD and 30 KD, respectively. Fractions of the main peak were pooled, were dialyzed and fractionally using liquid Express chromatography of proteins (FKLC) on LEAE/5PW-column using a gradient of NaCl. The active material was suirable when 0,11 M NaCl with FRLC-column. Peak fractions were pooled and subjected to VRGH using prepreparation C18-column and a gradient of ammonium acetate-n-propanol. After elution of 30% propanol with prepreparation C18-column active material was diluted (1:1) and again chromatographically using anal is momu peak P. O. at 280 nm elution profile. Similar results were obtained using a C4 column using H2Oh and acetonitrile containing 0.1% TFA as solvent, N-terminal amino acid sequence was determined by amino acid analyzer PTH, online Applied Biosistev 477 (Hewick and others , 1961) and a modified method of Stanley and Giklert (1981). Briefly, the labeling reaction was performed using 200 nm KL, 2 nm of chloramine-T, 10% dimethylsulfoxide, and 0.02% polyethylene glycol (total volume 25 μl) of 0.25 M phosphate buffer (pH 6.5). The reaction was carried out for 2 minutes atoC and stopped by adding 2 nm cysteine and 4 μm KL. Then KL was isolated from environments that do not contain Nal by gel filtration on ko-PD10-column (Pharmacia). Audirovannyj KL kept up to 2 weeks at 4oC.

Analysis of the binding

Buffer to link contained environment RPM1 1640 with 5% BSA (Sigma), 20 mm NERA (pH 7.5) and NaNO3. Experiments on the binding of unattached cells was performed in 96-Lucene tablets for cultivation density 2105cells per well in a volume of 100 μl. Experiments on the binding of 2-cells was carried out in hole 24 tablets in volume of 300 ál. Before adding the competitor ie before adding125I-KL for 30 minutes in 10-fold excess) was added unlabeled KL or rabbit serum against c-kit. Cells were incubated with125I-KL for 90 minutes and unattached cells were besieged by using 150 µl of FCS. Cellular debris was frozen and counted.

Immunoprecipitate and cross-mixing

VMS incubated with125I-KL in terms of binding and washed in FCS, and then in PBS at 4oC. the Cells were literally, as described previously [35] in 1% Triton X-100, 20 mm Tris (pH 7.4), 150= mm NaCl, 20 mm EDTA, 10% glycerol and protease inhibitors of phenylmethylsulfonyl (1 mm) and leupeptin (20 µg/ml). Lysates were subjected to thus with normal rabbit serum or with c-kit-specific serum produced by immunization of rabbits with a fragment of the domain of the tyrosine kinase v-kit ]23]; or with murine c-kit expressed from cDNA in the recombinant vaccinia virus [36]. For the experiments on co-precipitation of immunoprecipitate three times washed with washing And with 0.1% Triton X-100, 20 mm Tris (pH 7.4), 150 mm NaCl, 10% glycerol) was aluminiowe in the buffer for LTO-sample and analyzed using LTO-electrophoresis in SDS page and autoradiography. For the experiments on cross-shivani in PBS and literally, as explained above. After the deposition was carried out by washing under the following conditions: once in the wash (50 mm Trias, 500 mm NaCI, 5 mm EDTA, 0.2% Triton X-100), three times washing With 50 mm Tris, 150 mm NaCI, 0.1% Triton X-100, 0,1% Dsnb 5mm EDTA); and once in wash D (10 mm Tris, 0.1% Triton X-100).

Synthesis of cDNA, RCP-amplification (RT2PCR), and sequencing

RT-PCR amplification was carried out mainly by the method described in [53]. For cDNA synthesis, 1 µg row(A)-RNA from fused Balb/c T cells in 25 μl of 0.05 M Tris-Hcl (pH 8.3), 0,075 M KS1, 3 mm MgCl2, 10 mm dithiothreitol, 200 μm dNTP, and 25% araneina (Promega) were incubated with 50 PM antisense primer, and 50 units of reverse transcriptase of the virus murine leukemia, molony at 40oC for 30 minutes. Then added another 50 units of reverse transcriptase and continued to incubate for another 30 minutes, cDNA amplified first by bringing the reaction volume to 50 μl with 25 μl of 50 mm KC1, 10 mm Tris-HCl (pH 8.3), 1.5 mm MgCl20,01% (wt./about.) gelatin, 200 μm dNTP, and then by adding 50 PM sense primer and 2.5 units of DNA polymerase Tag with the following amplificatoare for 25-30 cycles in an automatic thermoacetica (Perkin-Elmer Cetus). Amplificatoare fragments were purified by electrophoresis on for sequence analysis [49].

The selection of cDNA and sequencing

In the present work used-a-DNA library fibroblasts T mouse, obtained from Clontech. The screening was carried out in duplicate and use Yscherichia coli

In 1090 as a bacterial host [48], and labeled at the 5'-end of the oligonucleotide as a probe. Hybridization was performed in 6SSC at the 63oWith, and the final wash of the filters was performed in 2SSC, 0,2% - ordinator at the 63oC. Recombinant phage was digested CoRI-enzyme and the insert was subcloned into M13 for sequence analysis. The nucleotide sequence of these cDNA on both strands and the overlap was determined by the method of dideoxy-termination of Singer [49], using as primers synthetic oligonucleotides (17 measures).

DNA and RNA analysis

End fragments were obtained genomic DNA, which was then digested restricteduse enzymes were fractionally by electrophoresis and transferred to nylon membranes. As probes for hybridization were used 1,4 kV KL cDNA and TIS-Dra/Sal (probe derived from the transgenic insertion site in the transgenic line TG. EV [85]).

Cells alb/T homogenized in isothiocyanate guanidine and in accordance with the method Seidna gel and transferred to nylon membranes (Nytran, Schlelcher Schuell), and then carried out prehybridization and hybridization in accordance with known procedures [86, 35]. As a probe for hybridization was used to 1,4 KL cDNA, labeled32P phosphate.

Obtaining monoclonal tel proteins c-kit-ligand

For selection of monoclonal antibodies to mice alb/s at the age of 8 weeks was intraperitoneally injected with 50 micrograms of purified soluble polypeptide c-kit-ligand (KL) of the present invention or its fragment (obtained as described above) in complete Freund's adjuvant (1:1 by volume). Then at intervals of 1 month mice were administered a booster injection (secondary injection) soluble polypeptide ligand or its soluble fragment, mixed with incomplete adjuvant-blockers, after which the mice took the blood through the tail vein. 4, 3 and 2 days before the merge cells to mice intravenously injected with a booster injection of 50 μg of the polypeptide or its fragment in physiological solution. Then carried out the merger of splenocytes with non-secretory myeloma cells according to standard procedures. Two weeks later, the hybridoma supernatant was skanirovali for binding activity against c-kit receptor protein as described above. Pological against c-kit-receptor used method, described above, except that mice were administered an injection and a booster injection of C-kit receptor protein.

Alternatively, to highlight the mouse monoclonal antibody to rat Sprague-Dawley or Louis was injectively murine polypeptide and the resulting splenocyte was hybridisable by fusion with myeloma cells of the rat (y3-Ag 1.2.3).

The results of experiments

Isolation and characterization of a mouse cDNA encoding the hematopoietic growth factor KL

KL-protein was purified from sulphur, air-conditioned cells Balb/c T, using a series of chromatographic steps, including aminobenzyl and reversed-phase chromatography high resolution, as described previously [37]. As mentioned previously determined N-terminal posledovatelnosti (40 amino acids) KL, which consisted of:

KEIXGNPVTDNVKDITKLVANPLBDYMITLNYVAGMXVLP

To obtain a nondegenerate homologous probe for hybridization was synthesized (as shown in Fig. 8) fully degenerate oligonucleotide primers corresponding to amino acids 10-16 (sense primer) and amino acids 31-36 (antisense primer) and having at their 5'-ends of the recognition sites of the enzyme, cDNA corresponding to KL mRNA sequences coding for RT-PCR). In cDNA synthesis and PCR amplification as matrix used poly(A)+-RNA from Balb/c 3T3 cells in combination with degenerate oligonucleotide primers.

Amplificatory DNA fragment was subcloned into M13 and sequenced for three inserts. It was found that the sequence between the primers are unique and encode the desired amino acid sequentially (Fig.8) Oligonucleotide (49 nucleotides) corresponding to a unique sequence of PCR products was then used for screening libraries gtlll-mouse fibroblasts. Was obtained of 1.4 kb, which, in its 3'-half, encodes an open reading frame extending up to the 3'-end of the clone, and 270 amino acids (Fig. 11). The first 25 amino acids KL sequences had the characteristics of a signal sequence. From purified protein (amino acids lasts 26 to 65, to the following signal sequence) was obtained N-terminal peptide sequence. The hydrophobic sequence of 21 amino acids (residues 217-237), which at its carboxyl end followed by positively charged amino acids, has characteristics of a transmembrane segment In the sequence, located between the signal paspolozheniya cysteine spacers. For the transmembrane segment, not reaching signal termination (the end of the clone), should the C-terminal segment of 33 amino acids. Therefore, the amino acid sequence of KL are the signs of a transmembrane protein: N-terminal signal peptide, extracellular domain and C-terminal intracellular segment.

To identify KL-specific mRNA in Balb/c T-cells was blot analysis RNA. (Fig.12). Using 1,4-kV-KI-cDNA as a probe the blots containing poly (A)+RNA identified a major transcript of 6.5 kV and two minor transcripts from the 4.65 kV and 3.5 square using labeled on the end of the oligonucleotide, derived from N-terminal protein sequence, were found identical transcripts. Then it was found, this result indicates that KL is encoded by the major mRNA, which in large quantities is expressed in cells of Balb/c 3T3.

The soluble form of KL is a ligand of c-kit-receptor

It has been shown that hematopoietic growth factor KL, originating from fibroblasts, stimulates the proliferation of exact primary bone marrow cells and peritoneal mast cells and promotes the formation of burst - centers differenzierung early at the hands of Eritrean is to istrirovali specific binding KL cells, expressing high levels of c-kit protein, namely, fat cells (VMS) and NIH 2-cells expressing C-kit cDNA. Using a modified chloraminated method [88] performed 125I-tagging KI to obtain high specific activity. The analysis, conducted by LTOs-electrophoresis in RAAG revealed a single band (28-30 KD) (Fig. 13); and the analysis on the proliferation of mast cells showed that the labeled material retained its biological activity. Then evaluated the binding of elevated concentrations 125I-KL with N1H-2 cells expressing C-kit cDNA, N1H 2-control cells, with normal VMS, and with W/W-6 W/+and Wv/Wv- VMS (4oC). The results illustrated in Fig. 14, showed that labeled KL associated with N1H 2-cells expressing C-kit cDNA and with (+/+)-6 (W/+)- and (Wv/Wv) fat cells, but is not associated control N1H 2-cells or fat (w/W)-cells. Wv-Mutation is the result of mizzensail in the kinase domain of C-kit, which reduces in vitro the activity of the kinase, but does not affect the expression of C-kit protein on the cell surface [36]. In contrast, W-mutation occurs as a result of delikli due to a violation of splashiness does not occur [36]. In addition, the binding 125I-KL can be completed with unlabeled KL and with two different antisera against c-kit. The results illustrated the binding125I-labeled KL with cells expressing C-kit on their surface.

To obtain direct evidence that KL is a ligand of C-kit-receptor, an experiment was conducted to determine whether the purified complexes "receptor-ligand" by thus serum against C-kit. To implement this experiment requires complex KL-C-kit was stable and was not exposed to detergents used to solubilize the C-kit receptor. Suitable for this purpose are the receptor-ligand complexes, derived from the closely related factor receptor activation colonies of macrophages (CSF-1) and system PDCF-receptors [89] . At incubation at 4oWITH125I-KL by washing the cells were solubilizers by lysis using Triton X-100 and precipitated with rabbit sera against v-kit and against C-kit, conjugated with protein a-Separate.125I-KL remained in immunoprecipitate obtained by incubation with anti-kit serum, but not with lack of contrebia demonstrated the highlight of the whole125I-KL from samples containing immune complexes obtained with anti-kit serum.

For further characterization of complexes of C-kit-KL-receptor-ligand", an experiment was conducted to determine whether cross-KL contact with c-kit. For this VMS incubated with125I-KL, washed and processed cross-stitched disuccinimidyl substrate. Then the cellular ligase was thus subjected to anticorodal against v-kit and analyzed using LTO-electrophoresis in SDS page. The autoradiograph revealed three patterns, one of which (approximately 30 KD) was a KL, precipitated together with the absence of cross-linking with c-kit; the other (180-190 KD) corresponded covalently linked c-kit-KL-moominmamma complex; the third macromolecular structure, which was on the boundary between separating and concentrating gels (Fig. 15V). The molecular structure of similar size was observed when allocating directly to LTO-page without prior thus. After precipitation with non-immune serum125I-labeled molecules were observed. The formation of macromolecular structures depends on incubated KL with fat cells is clearly to say, that KL specifically binds to C-kit receptor is a ligand of C-kit.

Mapping KL with respect to the SI locus

To install, encoded Lee KL at the SI locus, conducted recombination analysis in order to determine the position of KL on the map in relation to the locus, which is directly connected with 1. This locus is the site of transgenic insertions in transgenic lines TGEB [85]. It was found that genomic sequences cloned from customers insertions on the map correspond to the distance 0,90,8 cm from the SI locus. So this website is the most well-known marker for SI.

For mapping KL with respect to the site transgenic insertions tested by interspecific mapping, where used crossbreeding mice C57B1L/6J mice species Mus spretus. This method has allowed to establish that the polymorphism of restriction fragments (PFLPS) for the cloned DNA is observed more often among mice of different species than among mice of different inbred laboratory strains [90]. The relationship between the 1.4 kb-KL-cDNA probe and TIS Dra/Sal (probe from customers transgenic insecurely) was analyzed by evaluating the degree of coincidence of the inheritance of their respective allele (C57BL/6J or M. spertus) is d Taol - restriction digestion. The results of this analysis of the relationship shown in Table 2. Only one recombinant was detected in 53 of the offspring. This corresponds to the percentage of recombination 1,91,9. Since this value is very close to the value of the genetic distance measured between site transgenic insertions and SI, then the obtained result confirms the suggestion that map, KL corresponds to the locus S1.

The degree of coincidence inheritance C57B1/6 (B6) or M. spretus (Sp) alleles in the offspring obtained by interspecific hybridization (see experimental procedures) was determined by estimating the KL-cDNA (12,4 KB) probe and Ti Dra/Sal (obtained from the website of transgenic insertions, which is directly connected to S1, see results). The percentage of recombination was calculated by the method of green (1981).

The locus identified by KL, also investigated in mice bearing the original SI-mutation [50]. For this purpose was based on the observation of polymorphic in inbred strains, and these observations were used for genotyping in SI during embryo development. C57B1/6J mice bearing SI-mutation stored in the strain Snew/FeJ, was obtained by crossing, and FI-offspring carrying the SI allele were subjected to intercross, i.e. stradivarimany, was anemic and homozygous in respect Snew/FeJ - derived RFLP at the site of transgene integration (Fig. 16). Nanamica mice were either heterozygous SII+ - mice or wild-type mice, and they were heterozygous for Snow/FeJ and C57BL/6J-derived polymorphism or homozygous for C57BL/6J-polymorphism, respectively. In the analysis of genomic DNA from SII+ and SIISI-mice using the 1.4 kb-KL-cDNA probe, no hybridization with DNA homozygous SIISI-mice was observed (Fig. 16). Hence we can conclude that the locus encoding KL-protein deleterows the SI mutation. This fact confirms the view that KL is the product of the SI gene.

Discussion experiments

Detection allelism between protooncogene C-kit - and the mouse W locus revealed the pleiotropic functions of C-kit receptor in embryonic development and in the adult animal. In addition, it defines the primary genetic system of transmembrane receptor tyrosine kinase in a mammal. Mutations in the SI locus and C-kit/W-locus affect the same target cells. On the basis of complementary and parallel properties of these mutations was suggested that C-kit receptor is encoded by the SI locus.

Conducted e is blowing. Based on available information about the function of C-kit-receptor was identified and purified the proposed ligand of C-kit-receptor, designated KL [37]. It was also illustrated by the specic binding of KL with the C-kit receptor, as evidenced by linking KL c cells expressing a functional C-kit receptor and the formation of a stable complex between KL and C-kit protein. Received KL-specific cDNA clones, and it was shown that KL has a definite position on the map relative to the SI locus on mouse chromosome 10. In addition, it was also demonstrated that KL-sequence delegated in the genome SI-mouse. Summarizing the obtained results, it can be argued that KL is encoded by the SI locus and is the ligand for C-kit-receptor, and this in turn provides a molecular rationale for the SI-defect.

Amino acid sequence deduced from the nucleotide sequence of KL-cDNA-clone, suggests that KL is synthesized as an integral transmembrane protein. Structural features of the primary product broadcast KL are therefore very similar to the structural features of CSF-1. CSF-1 is synthesized as a transmembrane molecule that processincoming to assume, similar to CSF-1 KL is also synthesized as a protein on the cell surface that can be procession with the formation of soluble protein. A protein isolated from medium conditioned by cells of Balb/c 3T3, can then be obtained in a soluble form of KL by release from the membrane form by proteolytic release. Although posttranslational processing and expression of KL-protein has not yet been precisely characterized, however, KL-form associated with the cell surface, can mediate intercellular interactions, as it is typical for prospective proliferative and transport functions of C-kit/W-receptor system. In accordance with the expressed approval relative to the membrane - associated forms of KL, it was shown that a hybrid protein is obtained by merging the soluble C-kit receptor and alkaline phosphatase, associated with cell surface Balb/c 3T3 cells, but not with fibroblasts originating from SIISI-mice.

The most significant aspect of the identification of the ligand of C-kit-receptor is the fact that due to such identification is facilitated by the study of the pleiotropic functions of C-kit. It was also concluded that in the hematopoietic system-kit-mutati is compartmental stem cells, C-kit/KL play an important role in the maturation of erythroid cells, as evidenced by anemia observed in mutant animals. In addition, there has been registered a decrease in the number of CFU-E in embryonic liver W/W and SIISId-animals, but the number of BFU-E remained normal, resulting in can be concluded that C-kit/KL promotes differentiation from stage BFU-E to the stage of CFU-E [90, 35]. Accordingly, it was shown that KL stimulates proliferation and differentiation of BFU-E (on day 7), and also promotes the formation in the bone marrow of early erythroid multipotent predecessors, which appear in the culture at a later time (14-20) [37].

Based on the fact that the W and SI-mutant mice lacking mast cells, it was concluded that C-kit/KL play a major role in proliferation, differentiation and/or survival of the fat cells in vivo [72, 73]. However, at what stage of differentiation of fat cells requires the involvement of C-kit/KL, while still not exactly known. In vitro-education VMS from bone marrow or fetal liver does not require the presence of C-kit/KL because VMS can be with the same efficiency biogas produced from normal and from W-mutant mice [60]. Authors on the school fabric, in response to KL that pointed to a role of C-kit/KL in various stages of proliferation and differentiation of fat cells regardless of the presence or absence of IL3 and IL-4, which are mediators of inflammatory and allergic reactions [66]. In compartmental stem cells susceptible populations, obviously, are colony-forming unit spleen (CFU-S), which produce myeloid colonies in the spleen of mice irradiated with a lethal dose, and cells with long-lived repopulation suitable for differentiation of various cells[80, 81, 82, 83]. While it would be interesting to install, does KL on the ability of self-renewal or differentiation of hematopoietic stem cell populations in vitro, possibly in combination with other hematopoietic growth factors. Such information would help to accurately determine the stage of compartmental stem cells, which requires the participation of C-kit/KL. Another possible function of C-kit is, obviously, the ability to ease the transition of cells from the rest phase in cycleroad phase [31] . Increased sensitivity to irradiation at SIISIdand W/Wvmice may indicate such a function in the dynamics of stem cells; and, in addition, it is known that the genus is Tvout on the proliferation and survival of germ cells and their migration from splashplay yolk SAC to the genital ridges during early development. At postnatal gametogenesis C-kit expression was observed in immature and Mature oocytes and in spermatogonia a and b, as well as in the interstitial tissue [39]. When melanogenesis C-kit/KL, obviously, plays a role in proliferation and migration of melanoblasts from the neural scallop in the peripheral region during early development and in Mature melanocytes. Using the above information regarding KL can greatly facilitate in vitro studies of the function of C-kit receptor in these cell systems.

The area in which the function of C-kit-expressing cells is disturbed, and in particular the SI-mutant mice, is the area where is produced by C-kit-ligand. Due to the alien nature of the mutation precise definition of the types of cells that produce KL in vivo, have not been made yet. However, in vitro systems that produce genetic defect W and SI mutations shed some light on this question. In long-lived systems, cultures of bone marrow adherent SIISIdcells are defective, whereas non-stick hematopoietic cells are not defective; and systems co-cultures of mast cells and fibroblasts, SIISIdfibroblasts are defective, then, as the fat cells is the hematopoietic stromal cells, and embryonic fibroblasts and fibroblasts of the connective tissue produce KL. Cell line Balb/C T, which is of embryonic origin, Express significant levels of KL, which were used as a source for cleaning. The information obtained about the type KL-expressing cells can help in the evaluation of the function of C-kit in gastrointestinal tract, nervous system, placenta, and in some craniofacial areas, i.e. in areas where there was documented C-kit expression [35, 39]. It is known that neither SI nor W-phenotypes are not associated with these cellular systems.

To establish close links between KL-genome SI locus and locus transgenic insertions Tg. EB on mouse chromosome 10 was used interspecific backcross. This method was previously used for mapping Tg. S-locus near SI. The discovery of this fact that KL-coding sequences are delegated to the original 1-allele, once again confirms the identity of the SI locus with KL-genome. The size of the deletions in SI-allele currently not installed. However, it would be important to establish, does this impact on neighboring genes.

No KL-coding the placenta is about homozygous for the SI-allele mice die at embryonic period from macrocytic anemia, and rare surviving individuals absent pigmentation of the skin and no germ cells [3]. This phenotype similar to the phenotype of strong mutation with loss of function of C-kit/W, which indicates the ligand-receptor interaction of KL and C-kit. Although SIISI - W/W-homozygotes there are differences, for example, in the development of germ cells, however, the SI may have a more pronounced effect, and in haematopoiesis SI may cause more severe anemia; but it is not known whether these differences result from different genetic backgrounds of the strains, or they are possibly a consequence of the SI-deletions on neighboring genes [5].

The original W-mutation is an example-kit-null mutation (36). (Wl+) mice, heterozygous for the normal allele, has spots on the abdomen, but do not have a dim color, and normal hematopoiesis and gametogenesis. Weak phenotype of heterozygous W1+mice is a contrast to the phenotype of heterozygous SII+mice, which have moderate macrocytic anapau, a diffuse color skins (in addition to abdominal spots) and reduced in size gonad. Thus, a 50% dose KL gene is limiting, but not sufficient for normal function of C-kit-receptor,it-receptor plays a role in populations of immature cleto predecessors, as well as in more Mature cells during hematopoiesis, gametogenesis and melanogenesis. Strong SI - or W-mutations can block the process of differentiation of these cells, and therefore, the role of C-kit receptor in populations of more Mature cells remains unclear. SI and W mutations, in which C-kit/KL-function is only partially broken, often find their effect in more Mature populations of cells. There are many weak WI-alleles. Their phenotypes, such as gametogenesis and melanogenesis, can be of great value in identifying pleiotropic functions of C-kit-receptor system.

Experiment 3. KL-1 and KL-2

Experimental materials

Mouse and tissue culture

MousewBB6+/+, C57B1/6J and 129/Sv-SId/+ were obtained from the Jackson laboratory (the Jackson Laboratory, Bar Harbon, ME) [52], 129/Sv-SId/+ males and females were crossed and then obtained a 14-day embryos used for producing embryonic fibroblasts method Todaro and Green [54]. Mast cells derived from bone marrow adult + / + mice, were cultured in medium RPMI-1640, supplemented with 10% fetal calf serum (FCS); the environment, air-conditioned ER-3 cells; nonessential amino acids: sodium pyruvate and 2-mercaptoethanol (complete RPMI (C) [36]. Cells Balb/tenney 10% calf serum (CS), penicillin and streptomycin, COS-1 cells [18] were obtained from Dr. Jerrard Huewitz (SKI) and cultured in DME supplemented with 10% fetal bovine serum, glutamine, penicillin and streptomycin.

Production of antibodies against KL

Murine KL was isolated from medium conditioned by cells alb 3T3 using the analysis on the proliferation of fat cells, as described in the literature [37]. To generate antibodies against KL rabbits were immunized by subcutaneous injection with 1 mg of KL in complete Freund's adjuvant. Three weeks later the rabbit percutaneous has introduced a booster injection of 1 μg in incomplete Freund's adjuvant. Serum was collected one week, and then every two weeks. The commonly used125I-labeled KL has itinerary chloraminated method Stanley Gilbetr, modified as described previously [38].

Screening cDNA libraries

Row(A)RNA obtained from total RNA of fibroblasts from Balb/c T-cells by chromatography on oligo(dT) cellulose. Then prepared made in the usual way of plasmid cDNA (Invitrogen Inc). Mostly double-stranded cDNA was synthesized by oligo dT-priming and randomized priming. Nepalindromnoi BstXI-linkers were made with a blunt at both ends of the cDNA and after perevarivaniya then used for transformation b1061/P3 by electroporation, as a result, we generated plasmid library. The initial size of the library was approximately 107independent colonies. For screening this plasmid library was used marked the end of the oligonucleotide probe described previously [38]. Hydridization conducted in 6SSC at the 63oWith, and the final wash of the filters was carried out in 2SSC and 0.2% LTOs at the 63oC. Inserts of recombinant plasmids were isolated by digestion with HindIII and Hwa-enzymes, and then was subcloned into the phage plasmid MMR for further analysis.

R-amplification (RT-R) and sequencing

From tissues and cell lines were obtained total RNA by centrifuging in the isocyanate guanidine /CSCI (Chirgwin) [10]. RT-PCR amplification was carried out basically as described previously [38]. In this process we used the following primers:

Primer 1: 5'-CCCCAGCTTCGGTGCCTTTCCTTATG-3' (n. 94-107);

Primer 2: 5'-AGTATCTTAGAATTTCACCTCGAAATTCTC-3' (n.907-929);

Primer 3: 5'-CATTTATCTAGAAAACATGAACTGTTACCAGCC-3' (n. 963-978)

Primer 4: 5'-ACCCTCGAGGGTGAAATCTAAAYCTACTTG-3' (n. 1317-1333)

For cDNA synthesis, 10 μg of total RNA derived from cell lines or tissues, 50 μl of 0.05 mm Tris-HCl (pH 8.3), 0,75 Kl, 3mm MgCl2, 10 mm DTT, 200 μm dNTP, and 25% RENCASIA (BPL) were incubated with and at 37oC. cDNA was besieged by adding 1/10 volume of 3 M NaOH (pH 7.0) and 2.5 volume of absolute ethanol and resuspendable in 50 ml of ddH2O. PCR was carried out in 30 cycles in 100 μl of 10 mm Tris-Hcl (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, 0,01% (wt./about.) gelatin, 200 μm dNTP, 500 PM both sense and antisense primers, and 2.5 units Copolymers (Perkin-Elmer-Cetus) and Hind III sites and Xbal sites were inside sense and antisense primers, respectively. Amplificatoare DNA fragments were purified by electrophoresis on an agarose gel, digested with appropriate restricteduse enzymes and sublimirovanny in M13 mp18 and MMR for subsequent sequence analysis [49]. PCR products KL-1, KL-2, KL-S KL-SIddigested Hind III and Abal-enzymes and subcloned into expressing plasmid pCDM8 or pcDNAI (Invitrogen). Minipreparation plasmid DNA was obtained by the method of alkaline lysis [48] followed by extraction with phenol-chloroform, followed by precipitation with ethanol. Maxiprep plasmid DNA used for transfection of COS-1 cells were obtained using "Oiagen" - chromatography on columns.

The analysis to be protected from RNase

Ribsand for analysis of protection from RNase was obtained by linearization KL-1-containing plasmid RS DNAI using Spel Promega Gemini, Ribsand labeled to obtain highly specific activity, then hybridisable with 10 µg or 20 µg total RNA in the presence of 80% formamide overnight at 45oC. the Hybridization mixture was digested by RNase TI (Boehinger-Mannheium) and were treated with proteinase K [48], and then protected labeled RNA fragments were analyzed using 4% urea /polyacrylamide gel. Autoradiogram were analyzed by densitometry and part of the film was restored on the analyzer Phospholmage (Molecular Dynamics for the best resolution.

Transient expression of "KL"-cDNA in COS-1 cells

For transient expression of KL-cDNA in COS-1 cells were transfusional DEAE-dextranomer the method described previously [20], with minor modifications. Briefly, COS-1 cells were cultured to subconfluent state one day before use, was trypsinization and again were seeded on Petri dishes at a density of 6106cells on the Cup. After 24 hours the cells reached 70% of continuity, after which they were transfusional 5 micrograms of plasmid DNA in the presence of 10% DEAE-dextran (Sigma) in 6-12 hours. The medium containing the plasmid DNA was removed, and cells were subjected to chemical shocked with 10% DMSO/PBS++strictly within 1 Yserowany cells COS-1 were cultured in DME, supplemented with 10% fetal calf serum, 100 mg/ml, 100 mg/ml L-glutamine and antibiotics.

Pulse-chase and immunoprecipitation analysis "KE"-proteins

72 hours after transfection transfetsirovannyh COS-1 cells were used for pulse-chase experiments. These cells within 30 minutes, incubated with DME containing detalizirovannoi 10% fetal calf serum and containing no methionine; and then marked35S-methionine (NEN) at 0.5 μm/ml At the end of tagging environment for tagging was replaced with medium containing excess methionine. To assess the impact of phorbol-12-myristate-13-acetate (PMA) and A on proteolytic cleavage KL after changing the environment for tagging usual environment (at the end of tagging) to transfitsirovannykh the cells were added with 1 μm PMA or 1 μm And 23187. After a certain period of time cells and supernatant collected separately for immunoprecipitation analysis. Cell lysates were obtained as described previously [35] in a medium containing: 1% Triton-100, 20 mm Tris (pH 7.5), 150 mm NaCl, 20 mm EDTA, 10% glycerol, 1 mm protease inhibitors (phenylmethylsulfonyl), and 20 μg/ml lapatin. For immunoprecipitation analysis of KL-protein products used rabbit anticigarette FR is A (0.1% Triton-X-100, 20 mm Tris (pH 7.5), 150 mm NaCl, 10% glycerol). Conjugate anti-KL-whey-protein a-sepharose" were incubated with the supernatant and the cell lysate for at least 2 hours at 4oC. Then immunoprecipitate once washed in the Washing (50 mm Tris, 500 mm NaCl, 5 mm E TA, 0.2 Triton-X-100), three times Washing With 50 mm Tris, 500 mm NaCl, 0.1% Triton X-100, 0.1% of LTOs, 5 mm EDTA) and once in the Washing of 1 (10 mm Tris, 0.1% Triton X-100). For gel analysis immunoprecipitate was solubilizers in DSP-buffer for sample by boiling for 5 minutes and analyzed using DSP electrophoresis in SDS page (12%), and then spent the autoradiograph.

Evaluation of biological activity of soluble KL.

Mast cells derived from bone marrow of adult WBB6 +/+ mice, were cultured in the medium RPM1-1640, supplemented with 10% fetal calf serum, conditioned medium from WEH1-3B cells, nonessential amino acids, piracetam sodium, and 2-mercaptoethanol (RPM1-full environment), as described previously [37]. Non-stick cells were collected by centrifugation, weekly, nurtured and maintained at a density of <7 [13, 4]. Determined the effect of PMA treatment on biochemical characteristics of KL-1 and KL-2 in COS-1 cells. Pulse-chase experiments, about what eticos and that released KL-1 and KL-2 - protein products are virtually indistinguishable from those products, which were obtained in the absence of inducer. These results suggest that proteolytic cleavage of both KL-1 and KL-2 is activated by PMA in the same way. On the other hand/ this fact may indicate that PMA activates two different protease specific for KL-1 and KL-2, respectively; or alternatively, PMA activates only one protease, but at a very high level, which cleaves both KL-1 and KL-2, but with different speeds. Manory the restriction site in KL-1 contains, as expected based on the known C-terminal amino acid sequence of rat KL-amino acids PPVA ASSL (186-193) and may require an enzyme, such elastase [22, 34]. Based on the arguments made earlier, the sequence of site recognition in KL is, presumably, with the end from deletirovanie exon, and, therefore, may contain amino acids of REAKA (202-207) requiring enzyme, which by its specificity similar to KL-1-protease, or an alternative, which can be a trypsin-like protease. In addition, we determined the effect of calcium ionophore A on splitting KL. The fact that using this reagent was accelerated cleavage of KL-1 and KL-2, suggests that the prot is Fig. 22 ° C).

The biological activity of released KL-protein products.

To determine the biological activity of released KL-protein products supernatant transfected COS-1 cells were collected 72 hours after transfection and analyzed for activity in the test on the proliferation of fat cells. Fat cells, derived from bone marrow (VMMS), incubated for 24 hours with various dilutions of the collected supernatants and analyzed for the inclusion of3R-thymidine as described previously (Fig. 23). Supernatant produced from KL-1-transfectants were found to be 3-5 times more activity than the activity from KL-2-transfectants, which corresponds to the above-mentioned differences in the release of soluble KL from KL-1 and KL-2. It is important to note that proteins released from KL-1 and KL-2-transfectants, was found the same specific activity in the analysis on the proliferation of fat cells.

Steel dickie-allele resulting from deletion of the C-terminal KL-coding sequences, including the transmembrane and cytoplasmic domains.

Mouse homozygous for SId-allele are viable in contrast to mice on SI-Alleluia. Therefore, in these mice, c-kit-receptor system, obviously, has some residual activity. Since SIdthe mutation affects three lines of cell differentiation in a similar way, it can be assumed that this mutation affects the genetic properties of KL. Therefore, to study SIdat the molecular level KL-encoding sequence characteristics carried out first in this allele using technology PCR cloning. Using standard procedures received primary fibroblasts from SId/ + embryos. Then using RNA obtained from SId/+ embryonic fibroblasts and various primers amplified KL-coding region SIdtaking into account the possibility that SIdmay be a deletion mutation. By RT-PCR amplification using the full SId/+ - RNA and primers 1 and 2 were produced DNA fragment that migrated with a mobility identical to the mobility of the product obtained from RNA fibroblasts +/+; and after determining the sequence of this fragment was found that it is not different from the known KI-sequence. Therefore, it was concluded that this fragment represents the normal allele is as DNA fragment (850 and 1070 p. O.), obtained with primers 1+3 and 1+4, was subcloned into the PC M8, and then sequenced. IN KL-SId-cDNA was deleterows segment of nucleotides 660-902 sequence of the wild type, but instead was found to insert in a sequence of 67 p. O. (Fig. 17). As a result of these mutations, in the direction of 5' from the point deletions were introduced three amino acids of the termination codon. Predicted based on KL-SId-cDNA amino acid sequence consisted of amino acids 1-205 known KL-sequence + three additional amino acids (Fig. 17 and 19). KL-SId-amino acid sequence contains all 4 sites of N-glycosylation and all sequences contained in the soluble form of KL, but the transmembrane and cytoplasmic domains KL-1 wild type are deleteregvalue. Hence we can conclude that KL-SId-protein product is secretively protein having biological activity.

Biochemical characteristics and biological activity of KL-SId- and KL-S-protein products.

For comparison with KL-SId-protein product was designed truncated variant KJL-1, denoted by KL-S, which was introduced termination codon in the floor of the KL-containing plasmid and performed pulse-chase experiments in order to determine the biochemical characteristics of these two protein products. KL-SId-protein product quickly processionals, probably due to glycosylation and stood out in the environment, where after a 30 minute chase period were found majeure 30 KD species and with time their number was increased (Fig. 24). Biosynthetic characteristics of KL-S-protein products were very

similar to the characteristics of KL/SId(Fig. 24). And in this case, with time, the amount secreted in the medium of the material increased in contrast to cell-associated KL-S-protein product, the amount of which decreased with time.

To assess the biological activity of secreted KL-SId-and KL-S-protein products was carried out by analysis on the proliferation of fat cells. The medium from transfected COS-1 cells were collected 72 hours after transfection, and then various cultivation were used for analysis of proliferative activity against VMS in the absence of IL-3. Both samples were found significant biological activity, which to some extent was greater than activity KL-I (Fig. 23). The results convincingly demonstrated that KL-SId

Demonstration of allelism between c-kit and the mouse w locus sheds light on pleiotropy functions of c-kit-receptor during the development of the animal organism and the functioning of an adult organism, and also facilitates the identification of the ligand for this receptor (KL). In addition, a newly discovered allelism between KL and mouse-Steel-locus provides a molecular rationale for the relationship between w and SI mutations, which previously suggested by experts in mouse genetics on the basis of similarity and complementarity of the phenotypes of these mutations. Predicted transmembrane structure of KL leads to the conclusion that both forms, both membrane and soluble KL play an important role in the functioning of c-kit. In this application, this assumption is confirmed experimentally.

Firstly, it was shown that the soluble form of KL is produced by efficient proteolytic cleavage of the transmembrane precursor of KL-1. Secondly, it was shown that alternative splanirovany option KL-1, KL-2, in which the main site of proteolytic cleavage removed by splicing, also produces a soluble, biologically active form KL, albeit with a slightly lower efficiency. Thirdly, the concentrated, the expression of KL-1 and KL-2 in cells is tissue-specific. It was also shown that viable SId-mutation is the result of deletions, which includes the end of the KL-coding sequence, including the transmembrane domain, generating biologically active secreterial form KL. In addition, the phenotype of mice carrying SIdallele confirms the assumption made above about the role Sekretareva and membrane forms of KL in c-kit function.

On the basis of the close evolutionary relationship between c-kit and CSF-IR can be assumed that a certain relationship between the growth factors KL and CSF-1 exists in respect of their structure and topology. It is known that alternative splanirowannya forms of CSF-1-MRK encode protein products that differ in the sequences of the N-terminal transmembrane domain, spacer elements segment of 298 amino acids located between legenday part and the transmembrane domain of the protein [43]. In addition, alternative splanirowannya CSF-1-RNA transcripts differ in their 3'-untranslated regions [21] . Analysis of KL-PHK-transcripts in several tissues identified alternative playerwindow KL-RNA, which is the same as in the case of the notice, the expression of this alternative splanirovano RNA product flows through tissue-specific type. A recent comparative analysis of ligandin parts of KL and CSF-I revealed structural homology between the two proteins on the basis of limited amino acid homology comparison of the respective exons and the correct pairing of bases in the exon-kopirovani secondary structure [4]. In addition, the upper position of the 4-helical domains and cysteine residues, which form intermolecular disulfide bonds, implies the presence of related tertiary structures of domains of ligands KL and CSF-1; and the homology observed in the N-terminal signal peptides, transmembrane domains and intracellular domains of these two peptides may indicate that these domains are performing a similar function in these two proteins. These results confirm previous assumptions about the evolutionary relationships and structural homology between KL and CSF-I.

A distinctive feature of KL is that it had previously predicted a threefold structure of the transmembrane protein. Both forms of KL, KL-1 and KL-2 are synthesized as transmembrane proteins, which are processed by proteolytic cleavage system stage pressure in these two forms differ in their kinetics. Unlike KL-1 protein, where proteolytic cleavage runs very efficiently, KL-2 protein is more stable or resistant to proteolytic cleavage. Sequence encoded delegated by exon (amino acids 174-201) include With the end of the soluble KL-protein and the proposed site of proteolytic cleavage [27]. Therefore, in all probability, to generate soluble KL-2 protein of a second or alternative site of proteolytic cleavage, and cleavage can participate by another protease. The induction of proteolytic cleavage KL-1 and KL-2 in COS-1 cells activator of protein kinase C (PMA) and the calcium-ionophores A suggests that different types of cells, this process can take place on different types of regulation. Interestingly, soluble KL-2 protein has normal biological activity, which indicates that the sequence of encoded delegated exon, is not important for this activity.

On the one hand, in their membrane-associated variants of KL-1 and KL-2 can act as mediators of intercellular signal when the interaction; or, Altera KL are diffundere factors which can reach the target cells and its receptor on relatively short or longer distances. However, the soluble form of KL can also be associated with extracellular matricom, or they can be sequenced in this extracellular matrix is similar FCF, LIF or int-1 and, thus, to function for a short distance like membrane-associated forms [8, 33, 42]. In his cell-membrane form KL may be able to provide or maintain a high concentration of localized signal for interaction with the receptor-bearing target cells. In turn, the soluble form of KL can induce signal at low and variable concentrations. It is obvious that c-kit promotes cell proliferation, cell migration, survival of the cells and stimulates postmitotic functions in various systems of cells. Similarly, CSF-1-respitory systems the survival function of cells and cell migration may require a lower concentration factor than the function of cell proliferation [55]. Membrane-associated and soluble form of KL can serve different versions of c-kit function. The receptor for CSF-1 and c-kit can be adjusted according to the type of feedback through prefunctional the value of this process is still unknown, however, it has been suggested that the released extracellular domain of these receptors can neutralize CSF-1 and KL, respectively, in order modulation of these signals. In some cases, proteolytic cleavage leads to negative modulation of c-kit function, and therefore these processes can be considered as complementary or similar. In summary, it can be argued that the synthesis of variant membrane-associated KL-molecules and their proteolytic cleavage with the production of soluble forms of KL is important for the regulation or modulation of c-kit function in cells of different types in the process of development of the organism of an animal or organism functioning of an adult animal.

Characterization of SIdmutations at the molecular level represents a unique opportunity to assess the role of the soluble form of KL during development and in adult animals. SIdallele encodes the secretory variant of the KL protein (but not its membrane form) due to deletions, which includes the transmembrane domain and the C-end of KL. Therefore, the biological characteristics of SId/SIdand SI/ SIdmice should provide the key to identifying the role of soluble and membrane-mice are viable, but are characterized by severe macrocytic anemia, lack of fat cells in the tissue, the lack of pigmentation of the coat and futility. In most variants of this mutant phenotype of these mice is similar to w/wvvisible differences. Anemia in SI/SIdmice is obviously more susceptible to hypoxia than w/wvmice [46,47]. As gametogenesis, w/wvmice lacking the proliferation of primary germ cells and their migration slowed [32] . THE SI/SId-embryos, as well as w/wv-embryos, there is no proliferation of primary germ cells, however, the remaining cells retain the ability to properly migration, and, obviously, they reach the gonadal scallops in the corresponding period of development [29, 51]. Based on these experiments, we can conclude that SId-KL-protein is able to promote cell migration but not cell proliferation), and then to the formation of the membrane form KL, and therefore, it can play a decisive role in the proliferative response of c-kit. In addition, SI/SIdfibroblasts are unable to proliferation and maintenance of the fat cells of the bone marrow in the absence of IL-3 in contrast to normal embryonic fibral the SId-protein products, the inability of SI/SId-fibroblasts to maintain proliferation of fat cells, on the one hand, may mean that the amount of soluble KL-SIdprotein released by these cells is not sufficient to stimulate proliferation; but on the other hand, this phenomenon may indicate a critical role of cell-membrane form KL in the process.

KL in combination with IL-1, IL-3, G-CSF, GM-CSF

In normal murine cultures of bone marrow, we used murine KL (recombinant murine c-kit-ligand), and observed only a very small number of myeloid colonies stimulated one KL, however, the number and size of colonies was significantly increased by using combinations of KL and G-CSF, GM-CSF and IL-3 but not with M-CSF [103] . In HPP-CFC-analysis using bone marrow, obtained 24 hours after treatment of mice with 5-FU, there was an increase in colonies in combination with cytokines. The combination of KL with G-CSF, GM-CSF, IL-3, IL-7 or IL-6 are effective, and the combination of three or four factors are even more effective in stimulating HPP-CFC, and the most effective was the CSF or IL-3 in combination with IL-1, IL-6 and KL. In Fig. 25 shows HPP-CFC, stimulated by combinations of cytokine in kulturama as IL-1 + GM-CSF or IL-3 IL-1, was stimulated approximately the same number of HPP-CFC, as with combinations KL + IL-1 or KL + IL-3, but appeared to be particularly effective combination of three factors IL-1 + KL and either G-CSF or IL-3.

The Delta, or secondary CFU-analysis of early hematopoietic cells. Studies in mice.

The Delta analysis is a secondary count of clonogenic analysis, in which korotkozhivuscyego (7 days) suspension culture of bone marrow, devoid of the commutated predecessors and enriched early stem cells in the presence of various cytokines, research on the stimulation of survival, recruitment, differentiation and increase of stem cells and precursor cells. 5-FU-resistant stem cells were analyzed in primary HPP-CFC-analysis of multiple cytokine stimulation, as well as in the standard CFU-GM-analysis with the stimulation of only one CSF. In the secondary HPP-CFC and CFU-GM assays used suspension culture. Usually was determined by three parameters. The first parameter was the amplification linespecific restrictively predecessors-defined value representing the ratio of the total number of CFU-GM, susceptible to one type CSF (for example, G-CSF) in primary culture (input), m is the ratio of HPP-CFC-"entrance" to the total number of CFU-GM progenitor in the secondary analysis. Since CFU-GM, supposed to be descended from the earliest precursors, i.e. HPP-CFC, then this can serve as an indication of the differentiation of stem cells into precursor cells. And finally, the third parameter is the ratio NRR-CFC-login to the total number of secondary NRR-CFC. This option is the best indicator of self-renewal of stem cells, especially if HPP-CFC-stimulation in primary and secondary cultures due to the combination of IL-1, IL-3, and KL.

In the early stages of research (before using KL) different levels of increase in the number of CFC-GM susceptible to one type of CSF, and NRR-CFC-1 and 2 was observed in case of IL-1 was combined with M-CSF (20-30-fold increase), with G-CSF (50-100-fold increase), with a... (200-fold increase), while IL-3 and GM-CSF were produced limited degree of increase of progenitor cells, whereas M-CSF and GM-CSF not have produced such an increase. IL-6 was found less effective than IL-1 in synergistic action with M-CSF, GM-CSF or G-CSF, but with a combination of IL-3 and he showed the same efficiency as IL-1. The combination of IL-1 and IL-6 were detected additive and superadditive interaction with three CSF-views and IL-3. The presence of only one KL (obtained as described is the name "Mast Cell Growth Factor (growth Factor fat cells), published on 9 January 1992 and transferred to the Immunex Corporation, or, alternatively, obtained as described in European patent application 423980 titled "Stem Cell Factor (stem cell Factor), published on April 24, 1992 and passed A mgen Inc.) in phase suspension cultures caused a small increase in the production of progenitor cells (Fig. 26), but with a combination of KL with GM-CSF, IL-3 or IL-1 was observed 200-800-fold increase. More effective increase of progenitor cells gives the combination of IL-1, KL and either GM-CSF or IL-3, and with the combination of four factors such as IL-1 + KL + IL-6 or IL-3 or GM-CSF was observed up to 2500-fold increase in precursor cells. Counting generations of progenitor cells was performed based on the output of CFU-GM. HPP-CFC-login showed that the combination of the three factors (IL-1 + KL + IL-3 or CSF) was given relationship 6000-10000, and a combination of four factors (including IL-6) gave the relationship 8000-15000. As self-renewal, the generation of secondary HPP-CF-"input" with the combination of the two factors (KL with IL-1, IL-3 or CFU) reached values of 50-700, and when a combination of three factors (KL + IL-1 with IL-6, IL-3 or CSF) this value is reached values of 700-1300.

Experiment with fully differentiated cells produced in 7-on the radio. In this experiment, we measured the degree of self-renewal (using secondary HPP-CFC-generation) producing precursor cells (nitropropionate potential of CFU-GM) and morphologically identifiable differentiation of myeloid cells. The doubling time of the cell population required for the generation of these cells from a single predecessor, had reached the limits established for the rate of proliferation of mammalian cells. If these proliferation is supported by an earlier and even more rare cells than HPP-CFC-cage, you might have even shorter doubling time of a population. Reproduction of HPP-CFC in this Korotkova culture is unlikely to give a clear idea about the comparative increase in long-lived vosstanovleniya cells, and most generated HPP-CFC, probably represent a later stage in the hierarchy of stem cells. Analysis D12-CFU-S also showed an absolute increase in the number of cells in 7-day suspension culture with IL-1 + IL-3 or KL. Other researchers have shown that similar suspension cultures predecessors CFU-GEMM (probably Dolgorukaya regenerative stem cells also proliferate in the presence (IL-1 + IL-3) - the E. hematopoietic cells. Studies using human material.

It was shown that 4-HC treatment of human bone marrow leads to the elimination of the majority of progenitor cells capable of responding directly to GM-CSF by the formation of colonies in vitro, but are stored stem cells capable of hematopoietic recovery in connection with bone marrow transplantation. In preliminary studies on transplantation selection of CD34+also contributed to the increase in the number of bone marrow cells capable of long-term recovery. After a combined 4-NS-processing and selection of the 34+cells by immunocytometry education primary colonies in response to G-CSF or GM-CSF was negligible. However, using the 7-day suspension culture and subsequent secondary perchlorovinyl in GM-CSF has been shown that the impact on the treated bone marrow cells 7-day suspension in combination with IL-1 and IL-3 leads to a permanent generating the highest levels of secondary CFU-GM. IL-3 and IL-6 were not less effective than one IL-3, whereas other combinations of cytokines were significantly less effective. In this analysis the maximum stimulirovano was the most effective for stimulating the generation of progenitor cells.

The interaction between c-kit-ligand (KL) and IL-1, IL-6 and other hematopoietic factors.

In vivo desensitization of transplanted bone marrow by 5-FU is a simple way to increase the population of dormant hematopoietic precursor cells. Only one dose of 5-FU within 24 hours to reduce the number of early emerging CFU-S and more Mature CFU-C populations of more than 99%, while enriching the bone marrow more primitive predecessors. Later CFU-S are also sensitive to 5-FU-desensitization bone marrow (BM), which suggests that these cells are not the same as stem cells, responsible for the restoration of bone marrow (BM). On the contrary, it has been shown that BM-regenerating stem cells are resistant to the cytotoxic effect of 5-FU [105] . Brad ley and Hodgson, using 5-FU-desensitized bone marrow, identified the compartmentalization of progenitor cells HPP-CFC, which are able to form very large colonies in agar cultures.

The authors of the present application have investigated the interaction of IL-1, IL-6 and KL based on compartmental primitive murine progenitor cells [104]. Isolati factors taken both separately and in combination with CSF. The obtained results suggest that IL-1, IL-6 and KL in his early stimulation of hemopoiesis act in a unique way. This experiment using clonal cultures was then reinforced by subsequent analysis using short-lived liquid culture, i.e. analysis, which was described above. Was demonstrated by the ability of IL-1, IL-6 and KL handling.

Materials and methods

Mouse. Males and females (57 BL/6XDBA/2)F (B6D2F 1) mice were obtained from Jackson Laboratory (the Jackson Laboratory, Bar Harbor, ME). These mice kept in conventional conditions, giving acidified and/or autoclaved water for drinking. Individuals-hour, put together with the colony, was observed on specific pathogens. All mice were age at least 8 weeks.

Preparations of bone marrow and tissue culturing conditions.

Bone marrow (BM) from normal (NBM) or 5-FU-treated mice were obtained from the thighs, and sometimes the tibia from at least 3 mice in one experiment. Mice were treated by intravenous injection of 150 mg/kg 5-FU mg/kg in the amount of 150-250 ml Before cultivation VM twice washed by centrifugalized IMDM (Gibco, Grand Island, NY) supplemented with 20% FCS (Hyclone Laboratories Inc, Logan, UT) and 0.05% mg/HL gentamicin (Gibco). Counting cells in the bone marrow (BM) was performed using a Coulter counter model ZBI. (Coulter Electronics, Hialeah, FL). All used plastic utensils had a quality suitable for the cultivation of tissues.

Cytokines and antibodies.

Purified rhIL-I, sp act=1,32107units/mg (Syntex Laboratories, Inc.: Palo Alto, CA) was used at 100 units/ml of Partially purified and purified IL-6 were kindly provided by Steven Cillis (Immunex Corporation, Seattle, WA); partially purified IL-6 was used at 300 CESS units/ml, and purified IL-6 was used at 50 ng/ml of Purified KI received, as described in this application. Peeled rhG-CSF (Amgen Biological, Thousand Oaks, CA). Used 100 units/ml (sp act=H units/mg). Peeled rhM-CSF was used at 1000 units/ml (Immunex). Conditioned medium containing rmIL-3, were obtained from transient transfected COS-1 cells and like other growth factors used in the concentration required for maximum CFU-C stimulation. Rat monoclonal antibody against murine IL-6 was supplied from Genzyme (Cambridge, MA).

CFU-C assay.

LPP-CFC were analyzed in 35 mm Petri dishes containing 1 ml 5104NBM, suspended in culture medium, containing-the atmosphere with absolute humidity. HOO-CFC were analyzed using agarose system with dual layer described previously. 60 mm Petri dishes containing the bottom 2 ml layer consisting of culture medium, cytokines and 0.5% agarose, covered the top 1 mm thick 5-FU for 1-8 days before (dl - d8) 5-FU BM) analysis for HPP-CFC when the concentration of the cells 1103- 1105cells/culture. Double-layer culture were cultured for 12 days at 37oWith 5% CO2and 7% O2-the atmosphere when the absolute humidity. Cups were evaluated at low proliferative colonies containing at least 50 cells (LPP-CFC) and vysokopoligonalnye colonies with diameters constituting at least 0.5 mm (HPP-CFC). All CFU-C was calculated in the cultures of the three duplicates.

CFU-S-analysis.

Mice were irradiated 1250 Grams of gamma-source137Cs at a dose of approximately 90 gray per minute (G/min). Irradiation (1250 Grams) were single doses of 800 Grams and 450 Grams with an interval of 3 hours. Through 3-2 hours after the last exposure to mice intravenously were injected with BM cells. 12 days after transplantation of BM was calculated late CFU-S in the spleen, fixed in Bouin solution.

Delta--()-analysis.

The suspension culture was obtained, anxiety, and incubated for 7 days at 37oWith the presence of growth factors in 5% CO2-the atmosphere when the absolute humidity. After energy pipetting collected nephrolepsis cells from weak old cultures. Resuspendable BM cells from 4 duplicate-cultures was collected and 1 ml of culture was used to determine the saturation cell. The remaining 3 ml of cells were washed by centrifugation. The washed cells were analyzed on a secondary LPP-CFC, HPP-CFC and CFU-S. Secondary PP-CFC-sensitive G-CSF, GM-CSF and IL-3 were measured in 7-day CFU-S-cultures. Secondary HPP-CFC and LPP-CFC susceptible to IL-1 and IL-3, was evaluated after 12 days under conditions similar to the conditions described for the cultivation of HPP-CFC. Cells from cultures were diluted to 20-200-fold volume and to determine the number of secondary CFU-C. the Number of CFU-S present in the cultures after a week of cultivation, was determined by means of transplantation of mice 2-200-fold dilution of washed cells.

The fold increase in populations of precursor cells of the bone marrow in culture expressed in the units. The number of primary LPP-CFC, HPP-CFC and CFU-S present in the original (dl) 5-FU-BM-populations were measured in parallel suspension cultures. The Delta value was determined as the ratio of Pease on the depletion of adherent cells

Delta culture (12.5 ml 2,5105(dl) 5-FU-BM cells/ml) were placed in 25 cm2flask for tissue culture. Before cultivation VM freed from populations of adherent cells by a 4-hour incubation in culture medium at 30oC. non-stick cells transferred to the second 25-cm2the flask and both populations of cells were maintained under the conditions described above for the crops.

Analysis on the activity of cytokines.

Supernatant from cultures grown in 25 cm2flasks for tissue cultures were collected by centrifugation. Supernatant collected from cultures with (dl) 5-FU-BM (BM-cultures deprived of adherent cells, and BM cultures with adherent cells). IL-6 activity was measured using the analysis of cell proliferation using mouse hybridoma B9. The activity of cytokines was measured using a factor-dependent hematopoietic cell line NFS-60. The proliferation of NFS, % of cells in response to the activity of the growth factor was measured as described earlier.

The statistical analysis.

The significance of the obtained data was determined using bilateral t-test, Student.

Results

The activity of IL-6, IL-1 and KL in respect of the NBM.

The effects of IL-1, IL-6 and KL on 5-FU-BM.

The allocation of HPP-CFC and LPP-CFC from mouse (via 1-7 days after she was administered 5-FU) is shown in Fig. 1 and 3. There was a slight increase of the colonies in response to IL-1 and/or IL-6-stimulation, although it was constantly observed only for a few HPP-CFC and LPP-CFC. Linespecific CSF, G-CSF and M-CSF had a low ability to stimulate, whereas GM-CSF and IL-3 was found capable of being the fir factors.

Kit-ligand almost did not detect appreciable colony-stimulating activity, with an average were found only 1.3 HPP-CFC and 2.7 LPP-CFC, stimulated 1104d7 5-FU-BM cells (Fig. 30). The highest concentration of KL used in this study was 230 ng/ml and this concentration KL stimulates the formation vysokopolimernyh colonies in the presence of IL-1 and IL-6. At a concentration of 1 nm/ml of KL was observed in an average of 6.7 colonies, and at concentrations of KL from 10 to 100 ng/ml, the number of colonies reached a plateau in the range 120-147 HPP-CFC on 2,5104(d4) 5-FU-VMS-cells (data not shown). The addition of KL to G-CSF-containing cultures has led to an increase in the number of HPP-CFC in (d1) of 5-FU-BM, as well as to increase the number of LPP-CFC in both (dl d7) 5-FU-BM populations. Synergistic action of KL and G-CSF in stimulating HPP-CFC was especially pronounced in cultures (d4) 5-FU-BM (data not shown). Combination KL+M+CSF did not give any superadditive effect in the formation of colonies. However, KL had a strong synergistic effect in the presence of GM-CSF and IL-3 stimulation of HPP-CFC, IL-3 + KL combination was more effective in the formation of large colonies than IL-1 + IL-3 in both (d1 and d7) 5-FU-VMS-populations. The addition of KL to IL-3-containing cultures contributed 6-35-fold increase in the number of HPP-CFC in the whether IL-1 + IL-6-containing cultures has led to a dramatic synergistic action of these factors in promoting the growth of HPP-CFC (Fig. 30). The KL Association with IL-6 or IL-1 resulted in an average of 4.9 13.7 vysokopolimernyh colonies from 1105(d1) 5-FU-BM cells, respectively. In addition, in response to all three cytokines was observed education 42,0 HPP-CFC 1105cells. These results clearly demonstrated the existence of subpopulations of HPP-CFC, which for the formation of large colonies requires stimulation of IL-1, IL-6 + KL. Answer (d7) 5-FU-VMS on these combinations of growth factors was similar to the response (d1) of 5-FU-VMS. However, the number of HPP-CFC, stimulated by a combination of IL-1, IL-6 + KL (d7) 5-FU-BM was ten times less than the maximum number of HPP-CFC, which can be stimulated by adding GM-CSF to these three factors. The number of colonies of HPP-CFC, stimulated (d7) 5-FU-BM populations of four cytokines, only slightly different (a little more than twice) the number of colonies stimulated IL-1, IL-6 + KL.

The addition of IL-6 to cultures containing KL and CSF, did not give a significant increase in the formation of colonies over conventional additive effect, which could be expected from a combination of two factors IL+6, KL with CSF (Fig. 30). For example, the combination of IL-6, KL + GM-CSF leads to the formation of approximately 30 vysokopolimernyh colonies 1105(d1) 5-FU-VMS-cells. is x IL-6 + KL + GM-CSF (4 and 20 HPP-CFC, respectively), that suggests that the combination of these factors will not give any additional proliferation HPP-CFC.

In contrast to the above results, obtained using IL-6, the addition of IL-1 to cultures containing KL and CSF, did not give synergism (Fig. 30). This synergism was most pronounced in cultures (d7) 5-FU-BM cultured with combinations of IL-1, KL + G-CSF. The combination of any two factors of the three cytokines stimulated the formation of 5 or less NRR-CFC, whereas the combination of IL-1, KL + G-CSF stimulates an average of 100 HPP-CFC 1104BM cells. Synergistic effect, although less pronounced, was also observed when using IL-1, KL + GM-CSF or IL-1 stimulation (d7) 5-FU-BM. The same superadditive action, were also observed in (dl) 5-FU-BM-populations in the presence of combinations of IL-1, KL + G-CSF or M-CSF. The formation of a large number of HPP-CFC in (d1) of 5-FU-BM were stimulated with combinations of IL-1, KL + GM-CSF or IL-3, however, this effect is due to the additive effect of these growth factors in different populations of HPP-CFC.

As mentioned above, the greatest number of HPP-CFC was stimulated by the combination of four growth factors, the optimal number of colonies gave the combination of IL-1, IL-6, KL + GM-CSF-CSF or IL-3. positive colonies. Only using a mixture of IL-1, IL-6, KL + M-CSF was observed the increase of HPP-CFC, caused, apparently, by the synergy of all four factors in stimulating additional growth of large colonies, which was not observed with combinations of a smaller number of cytokines. The addition of IL-6 to the combination of the cytokines IL-1, KL + G-CSF, GM-CSF or IL-3 did not give superadditive effect. In most cases, the number vysokopolimernyh colonies stimulated IL-1, IL-6, KL + G-CSF, GM-CSF or IL-3 was not significantly exceed the number of HPP-CFC, stimulated with combinations of IL-1, KL + G-CSF, GM-CSF or IL-3.

The increase in 5-FU-BM-cultures.

The number of non-stick cells, isolated after 7 days of cultivation in crops, characterizes the type of response observed in clonal cultures of 5-FU-BM with different combinations of cytokines (Fig. 31). The control culture (dl) 5-FU-BM obtained without stimulation by cytokines, were found on average 39% decrease in the saturation of the cells with a predominant survival cell populations of monocytes/macrophages. The addition of IL-1, IL-6 or KL was not given to increase the number of cells above its original level. Except for a small increase in response to stimulation of GM-CSF and IL-3 these cultures were stimulated only with enablegui from crops, stimulated by combinations of UL-1, KL + GM-CSF or IL-3; and the subsequent addition of IL-6 to these cultures did not give a significant increase in the number of cells. The appearance of immature myeloid cells correlated with the observed proliferation of cultures. In one experiment, IL-3 stimulated cultures containing about 50% Mature segmented neutrophil granulocytes and miriagou, 25% of metamyelocytes, 20% of plasmic order has been revealed and 3% of blast cells. The percentage of blast cells was increased with the addition of IL-1 (22%), IL-6 (18%), KL (24%), IL-1 + IL-6 (12%), IL-1 + KL (51%), IL-6 + KL (43%) and IL-1, IL-6+KL (46%) of the IL-3-containing cultures, the greatest total number of blast cells (6,1105) were obtained from cultures stimulated with IL-1, KL, IL-3, which is about 200-fold increase compared with baseline (dl) 5-FU-BM-population.

Control cultures grown without added cytokines did not increase cell precursors LPP-CFC-populations compared with baseline values (Fig. 32). The addition of colony-stimulating factors G-CSF, M-CSF, GM-CSF and IL-3 gave a clear increase of cells (the average value was 3.4; 2,4; 23 and 140, respectively). One IL-1 stimulated more than 60-fold increase in LPP-CFC, and the Association of IL-1 with CSF resulted in synergistic expansion of LPP-CFC. N-3) + 63(IL-1)=203). IL-6 stimulated a small but significant increase in LPP-CFC (-value=3,4; p < 0,01). More than additive effect was observed using a combination of IL-6 + G-CSF and IL-6 + IL-3, KL did not give a significant increase in LPP-CFC-cultures (p=0,08). The combination of KL and CSF was given, however, more than additive effect in all cases. The combination of KL + IL-3 had the same efficiency as IL-1 + IL-3 to stimulate growth of LPP-CFC (mean value = 485 and 520, respectively; p=0.21). Delta culture, stimulated IL-1 + IL-6 in combination with CSF had higher-value in all cases, the cultures stimulated with IL-1 or IL-6. The increased expansion of LPP-CFC with additive effect stimulated all combinations of IL-1, IL-6 + CSF except for the combination of IL-1, IL-6 + M-CSF (-value= 300, and for IL-1 + M-CSF this value was 140, and for IL-6 + M-CSF-value=2,8). The combination of IL-6 + KL gave synergistic effect in stimulating LPP-CFC (more than 200 times), but adding these two cytokines to CSF-containing cultures led only to an additive increase in precursor cells. The combination of IL-1 and KL synergistically stimulated more than 1000-fold increase in LPP-CFC. Add G-CSF, GM-CSF or IL-3 to IL-1 + KL-containing cultures further increased the number of LPP-CFC (average value 1100, 1200 and 1400 CE is profiled combinations of IL-1, IL-6, KL + IL-3 were observed to 1800-fold increase in LPP-CFC despite this increase this value, the addition of IL-6 to IL-1 + KL-containing crops has not made a significant contribution to the observed increase in precursor cells (P > 0,05).

The increase of HPP-CFC in the Delta crops.

Experiments were carried out to assess the ability to stimulate HPP-CFC various combinations of cytokines (Fig. 33). As in the case of increase of LPP-CFC, the greatest increase in HPP-CFC was achieved in cultures stimulated with combinations of IL-1, KL + CSF, CSF, taken separately, stimulated only a very modest increase in HPP-CFC. IL-6 stimulated increase NRR-F, and IL-6 together with IL-3 was more effective in increasing HPP-CFC than one IL-3. In contrast to IL-6 IL-1 demonstrated synergism in combination with time four CSF. KL in combination with all cityrama CFC also found a more than additive effect in increasing HPP-CFC. The combination of IL-1 + IL-6 without CSF or together with SF was more effective than IL-1 or IL-6, taken separately. A particularly striking synergism was observed when using IL-1 + IL-6 in combination with M-CSF (average values were: IL-6 + M-CSF = 1,0; IL-1 + M-CSF = 13,2; and with IL-1 + IL-6 + CSF = 65,7). The addition of IL-1 or IL-6 K-cultures containing KL alone or in whom the e adding CSF cultures, containing KL or together with IL-2 or IL-6, did not give a significant increase in HPP-CFC. The greatest increase in HPP-CFC was observed in cultures stimulated by a combination of IL-1, IL-6 + KL (-value=705).

The production of secondary HPP-CFC in the cultures was assessed by using clonal analysis in the stimulation of IL-1 + IL-3 (Fig.33). Were also investigated other combinations of cytokines, such as IL-1 + GM-CSF or IL-1 + M-CSF for their ability to stimulate secondary HPP-CFC. Determination of the number of secondary HPP-CFC, cultured in the presence of IL-1 + M-CSF or GM-CSF, it was quite difficult due to too many secondary LPP-CFC in relation to the number of HPP-CFC, stimulated by these combinations of cytokines. Evaluated the effectiveness of IL-1 and KL as stimulating growth factors secondary HPP-CFC (Fig. 34). Unlike any other combinations of the studied cytokines, a sharp increase (IL-1 + KL)- sensitive precursor cells in culture was not observed, as it was recorded in case of increase (IL-1 + IL-3) - sensitive HPP-CFC and LPP-CFC.

The increase in CFU-S in crops.

For further characterization of populations of BM cells that appear in crops was investigated increase FC in response to STI increase in HPP-CFC and LPP-CFC, that are consistent with the results shown in Fig.32 and 33. In these cultures was also observed increase in CFU-S, which exceeded the increase in HPP-CFC. When stimulating combinations of IL-1 + IL-3 and IL-1 + KL, the number of late CFU-S was increased more than 100 times. These results were compared with the increase of HPP-CFC and CFU-which, as we know, occurred in mice treated in the 5-FU treatment; in vivo-increase (in vivo) was measured as the ratio of the total number of hip HPP-CFC, LPP-CFC and CFU-S in (d8) 5-FU-BM to the total number of colonies observed for 1 day (1d) after 5-FU-BM to the total number of colonies observed in 1 day (1d) after 5-FU treatment. In vivo increase of progenitor cells was similar to the increase in in vitro cultures, except that the increase in LPP-CFC in vivo was less than the increase observed in vitro.

Discussion.

These studies have illustrated the role of IL-1, IL-6 and KL as regulators of proliferation of primitive hematopoietic cells. These cytokines, taken separately, have a limited ability to stimulate proliferation of hematopoietic precursor cells in the clonal analysis of the cultures described in this application (Fig. 20-30). But IL-1, IL-6 and KL, taken together, reveal synergistic action with the authors of this proposal have been able to distinguish between populations of HPP-CFC and LPP-CFC, present in 5-FU-desensitized bone marrow (BM). The ability of IL-1, IL-6 and/or KL-regulation of colony-primitive hematopoietic cells was also confirmed by experiments using short-lived crops (dl) 5-FU-BM. -Analysis in order to measure the influx of populations of early precursors in response to stimulation by cytokines, showed that the largest increase in LPP-CFC and HPP-CFC depends on synergistic effects of IL-1, IL-6, KL and CSF on early hematopoietic precursor cells (Fig. 32-35).

An important role of IL-1 as a regulator of early haematopoiesis known since the time of its identification as a factor (Hemopoietin-1), which has synergistic activity present in the conditioned medium of cell lines carcinoma of the bladder 5637. In accordance with previously obtained results, the authors of this application showed that IL-1 have a synergistic effect in conjunction with G-CSF, M-CSF, IL-1 or KL during stimulation of HPP-CFC (Fig. 29 and 30). The ability of IL-I to stimulate the proliferation of primitive hematopoietic cells was also observed in the analysis (Fig. 31-33). Synergistic activity of IL-2 in combination with G-CSF, M-CSF, GM-CSF, IL-3 or KL was demonstrated by its ability stimulirovannykh studies give grounds to assume, the combination of the cytokines IL-1 + IL-3, G-CSF, M-CSF, GM-CSF. In crops, the combination of IL-1 + IL-3 can stimulate an increase in LPP-CFC and HPP-CFC in 520 and 83% respectively. This increase populations of progenitor cells was higher than the increase stimulated combinations of IL-1 + G-CSF, M-CSF or GM-CSF. However, the synergism observed between IL-1 and KL has a more effective catalytic effect on (dl) 5-FU-BM than IL-1 + IL-3.

The number of LPP-CFC in the Delta cultures stimulated with IL-1 + KL, has increased more than 1000 times, and the number of HPP-CFC - 280 times.

It was found that hematopoietic activity of IL-6 differs from the activity of IL-1. It was found that the combination of IL-6 + IL-3 or KL has a synergistic effect in stimulation of HPP-CFC (dl-d7) 5-FU-BM (Fig. 30). IL-6 and KL also act synergistically with the stimulation of CFU-C from NBM (Fig. 28). In the analysis was to illustrate the synergism between IL-6 and either IL-3 or KL and increase LPP-CFC and HPP-CFC (Fig. 5 and 6). In promoting HPP-CFC, IL-6 + IL-3 are not as effective as IL-1 + IL-3 (-size 40 and 83, respectively). It was found that the combination of the three factors IL-1, IL-6 and M-CSF have a synergistic stimulatory effect on the HPP-CFC (dl-d7) 5-FU-BM. In addition, the analysis also showed synergism of IL-1, IL-6 and M-CSF to increase LPP-CFC and HPP-CFC-populations. the highest growth HPP-CFC (-value = 705) in the cultures was observed when adding IL-1, IL-6 + KL to these cultures. This pattern of synergistic interaction between IL-1, IL-6 and KL in the regulation of pluripotent hematopoietic precursor cells.

Stimulating effect of KL on early hematopoietic precursor cells, illustrated in these studies corresponds to the activity, contributing to the growth of stem cells, which plays an important role in the cloning of the RL gene. Answer NBM-predecessors on the stimulatory effect of IL-1, IL-6, G-CSF, or IL-1 demonstrated synergism in combination with KL (Fig. 28). As previously reported, KL does not encourage kolonialapologie in response to M-CSF from NBM. A similar pattern of response was observed using 5-FU-BM; KL was found synergism in combination with IL-1, IL-6, G-CSF, GM-CSF or IL-3, but not with M-CSF (Fig. 30). Sharp synergic stimulation of HPP-CFC observed using a combination of IL-1 + KL, can be enhanced even more by adding CSF. The most striking synergism was observed with IL-1, KL and G-CSF in the culture from (d1 and d7) 5-FU-VMS. Optimal hematopoietic response was observed using a combination of the four cytokines IL-1, IL-6, KL and CSF. But only the combination of IL-1, IL-6, KL and M-CSF, had a synergistic stimulatory effect on HPP-CFC. The combination of IL-1, IL-6, KL and GM-CSF or IL-3 Stimorol Nkrumah (Fig. 31-33). These results illustrate the important role of KL in the regulation of cell proliferation early gemopoeticescoe cells.

HPP-CFC occupy a certain place in the hierarchy of cells that can be identified based on their needs of a growth factor and/or equipment of their physical separation. Identification of two early hematopoietic cells HPP-CFC-1 and HPP-CFC-2 correlates with the division of progenitor cells on the basis of their retention mitochondrial dye rhodamine-123. Weakly increased the rhodamine-123 cells are more primitive phase (HPP-CFC-1)cells for their proliferation require synergistic effects of IL-1, IL-3 and M-CSF, while the phase of HPP-CFC-2 cells does not require stimulating factor IL-1. The more primitive nature of IL-1 + CSF-stimulated early predecessors is supported by the observed synergistic effect of IL-1 + CSF to increase LPP-CFC and HPP-CFC in the analysis (Fig. 32 and 33). In addition, the regulation of primitive hematopoietic cells can be controlled by growth factors IL-6 and KL. The ability of IL-6 and KL to stimulate growth of HPP-CFC in culture, obviously, may indicate their role in the stimulation of the earliest precursors of HPP-CFC-1. These data confirm predpriyatii for their proliferation of multiple growth factors. The maturation of these early progenitor cells of Hpp-CFC-1 HPP-CFC-1 entails the restriction requirement stimulation of multiple cytokines for proliferation. The conclusion concerning the hierarchy of HPP-CFC confirms the observation of the fact that in response to the stimulation combinations of IL-1, IL-6, KL + GM + CSF of more than 3% (d7) 5-FU-VMS-cells are able to form HPP-CFC (Fig. 30), which is much higher than the expected frequency of occurrence multipotent stem cells in the bone marrow (BM).

The increase of HPP-CFC in crops, obviously, indicates expansion multipotent hematopoietic precursors. However, these post-HPP-CFC-culture in the hierarchy of HPP-CFC are not yet understood. The observed increase in late appearing CFU-S in-cultures confirms the existing opinion that the number multipotent hematopoietic precursor cells, increases in the conditions of the analysis (Fig. 35). In response to stimulation of the suspension cultures, purified and strongly or weakly stained with rhodamine-123 precursor cells, there was a 100-fold increase in the number of CFU-S. the Results obtained by the authors of this application, contrary to existing messages about reducing the CFU-S in liquid cultures (d2) 5-FU-BM-driven composition of IL-6 + IL-3 or KL, is concerned about the expansion of progenitor cells upon stimulation by cytokines IL-1, IL-3 or KL, can be successfully used in bone marrow transplantation, HPP-CFC susceptible to IL-1 + KL, found a minimal increase, influenced by a combination of IL-1, IL-3, IL-6 and KL-cultures (Fig. 34). The ability of IL-1 + KL to stimulate the growth of HPP-CFC from 5-FU-BM, as well as to stimulate a large increase in the number of early progenitor cells in the analysis, indicates that IL-1 + KL can affect the cellular pool or primitive multipotential precursor cells. Limited expansion (IL-1 + KL) sensitive HPP-CFC allows to make a conclusion about the limited ability of growth factors IL-1, IL-3, IL-6 and KL to stimulate the self-renewal of early hematopoietic precursors and stem cells in the analysis.

(IL-1 and KL)-induced proliferation and the effect of TGF and MIPI.

As previously reported, TGF and MIPI (inflammatory protein-I macrophages) are able to inhibit the growth of progenitor cells.

It has been suggested that any of these cytokines may act as a negative regulator of proliferation of hematopoietic stem cells, although both factors previously not directly compared in the analysis for the detection of stem cells. In the analysis of mouse NR-colonies of the uracil, and considered only colony with vysokopropulsivnym potential (0.5 mm). IL-1 and KL stimulate predominantly early hematopoietic precursor cells. Therefore, the authors of this proposal have been assessed exposure to TGF and MIPI proliferation NRR induced IL-1 and KL. Data obtained from two separate experiments (each performed in triple redundancy), expressed as the number NR-colonies induced by combinations of growth factors in relation to the number of colonies induced by only one GM-CSF (GSM) (see tab. 3).

These results showed that TGF "cancel" synergistic proliferation NRR colonies stimulated IL-1 and/or KL with GM-CSF, whereas VIPI such action is not. In addition, TNF/HPP-colonies, induced by IL-1 + KL, at that time, as MIPI actually stimulates the formation of colonies NRR in these conditions. Therefore, the authors of this proposal, it was concluded that TGF, but not MIPI, acts as a negative regulator investigated in the present work, the populations of hematopoietic progenitor cells. This is a very important conclusion can be taken into account in the design of protective chemotherapy.

The study of KL in combination with IL-3, EPO or GM-CSF in humans

BFU-E, stimulated by a combination of rhEPO+rmIL-3 and/or rhKL, were not detected in 6 patients with Fanconi syndrome with varying degrees of bone marrow failure. Myeloid colonies was not observed in 4 patients and were significantly reduced in 2 patients upon stimulation with either rhIL-3, or rhGM-CSF. These 2 patients were additionally introduced rmKL or rhIL-3, resulting in the average value of the frequency was increased. The third patient with DC rhIL-3 + rmKL induced myeloid colony, one of the Institute one rhIL-3 or rhGM-CSF or instead of rhEPO and rhIL-3 (13% of normal control). The last patient was an increase in the size and number of BFU-E, as well as increased hemoglobinuria adding rmKL, rhIL-3 or rhGM-CSF-induced myeloid colonies were slightly reduced, a KL induced a corresponding increase in the frequency of colonies.

The interaction between C-kit-ligand (KL), GM-CSF and tumor necrosis factor cells and the influence of these interactions on the development of pre-dendritic and dendritic cells in humans.

Dendritic cells are the most patentnymi antigen presenting cells, inducing the formation of primary specific T-cells in response to exposure to antigen in vivo and in vitro. Dendritic cells generated in vitro, can be used after exposure to the antigen for secondary immunization for vaccine therapy against HIV and tumors. The authors of this proposal have been developed in vitro system to generate people of dendritic cells from CD34+-population of bone marrow, peripheral and Central blood person. However, it was found that the presence of GM-CSF and TNF is required for the differentiation of dendritic cells in suspension culture and colonogenic analysis; and C-kit-ligand synergistically increases the number UP>+
-bone marrow cells of an adult for the development of dendritic cell colonies is absolutely necessary synergistic stimulation KL in combination with GM-SF and TNF. IN CD34+-populations of blood GM-CSF + TNF induce the formation of colonies of dendritic cells, but adding KL frequency of formation of colonies increased (table 1b). Comparison of several cytokines showed that the maximum formation of colonies of dendritic cells was stimulated by combinations of KL, GC-CSF and TNF (table 1C). In systems suspension cultures, the combination of IL-1 + KL + IK-3 contributed to a 100-fold increase pretending cells on day 14 of cultivation; and the addition of GM-CSF + KL + TNF contributed to the differentiation of these cells into dendritic antigenpresenting cells in connection with allogeneic reaction of mixed lymphocytes and mitogenesis CD3-T lymphocytes. KL has a unique stimulatory effect on the generation of pretending and dendritic cells to primitive precursor cells of the bone marrow/stem cells.

CD34+cells were isolated from managernew cellular fractions of normal bone marrow by immunomagnetic separation using microspheres for fractionation. IL-1, KL, IL-3 and GM-CSF apolloni dendritic cells.

CD34+cells were isolated by immunomagnetic separation, and were cultured at a density of 103cells/ml in 10 DM-medium, supplemented with 20% fetal calf serum and 0.36% agarose, within 14 days. After 14 days, colonies dendritic cells were identified by morphological analysis, rhKL, IL-3, and rhGM-CSF was used at 10 ng/mg

CD34+cells were isolated by immunomagnetic separation and cultured at a density of 103CL//ml, rhKL, rhIL-3, rhGM-CSF, rhG-CSF, rhM-CSF and rhPIXY used at a density of 10 ng/ml 14 days after culturing dendritic cells were identified by morphological analysis.

1. Pharmaceutical composition comprising (a) an effective amount of c-kit-ligand comprising a polypeptide, amino acid sequence which (i) is encoded by the nucleic acid sequence shown in Fig. 11A-11D (SEQ ID NO: 1), or (ii) the naturally occurring hematopoietic variant of the polypeptide (i); (b) an effective amount of hematopoietic factor, and (c) suitable pharmaceutical carrier.

2. The pharmaceutical composition under item 1, characterized in that is used to enhance bone marrow engraftment when transplanted to a mammal, is the rpm die, what is used to improve the recovery of bone marrow in the treatment of allazei bone marrow or mielosupression caused by radiation, chemicals or chemotherapy and hematopoietic factor is IL-1 or G-CSF, in an amount effective to improve the recovery of bone marrow from a mammal.

4. The pharmaceutical composition under item 1, characterized in that is used for the treatment of acute myeloid leukemia in a mammal, and one of gomeopaticheskih factor is GM-CSF or G-CSF, in an amount effective for the treatment of acute myeloid leukemia in a mammal.

5. The pharmaceutical composition under item 1, characterized in that is used for the treatment of chronic myeloid leukemia in a mammal, and one of the hematopoietic factor is GM-CSF or G-CSF, in an amount effective for the treatment of chronic myeloid leukemia in a mammal.

6. The pharmaceutical composition under item 1, characterized in that is used for the treatment of radiation in a mammal, and hematopoietic factor selected from the group consisting of G-CSF, GM-CSF or IL-3 in an amount effective for the treatment of radiation in a mammal.

8. The pharmaceutical composition according to p. 7, characterized in that one of the hematopoietic factor is IL-1, IL-3, IL-6, GM-CSF, G-CSF or PIXY.

9. The pharmaceutical composition according to p. 7, wherein the hematopoietic factors are (a) IL-1 and IL-6, (b) IL-1 and IL-3, (C) IL-1 and GM-CSF, (d) IL-1 and MIP1, (e) IL-1 and G-CSF, (f) IL-6 and G-CSF, (q) IL-3 and G-CSF, or (h) PIXY and G-CSF.

10. The pharmaceutical composition according to p. 1, wherein the hematopoietic factor is (a) IL-1, IL-6 and IL-3, (b) IL-1, IL-6 and GM-CSF, (C) IL-1, IL-3 and G-CSF, or (d) IL-1, IL-6 and G-CSF.

11. The pharmaceutical composition under item 1, characterized in that is used to increase the level of stem cells in the peripheral blood and hematopoietic factor is IL-1 or G-CSF, in an amount effective to cause the occurrence of stem cells in the peripheral blood.

12. The way to increase the level of stem cells in the peripheral blood of a mammal, comprising the administration to a mammal the pharmaceutical composition under item 1.

13. A nucleic acid molecule having the antisense sequence of the nucleotide sequence shown in Fig. 11A-11D (SEQ ID NO: 1), and capable of contacting the mRNA with the Kit-ligand and prevent its translation.

th blood ex-vivo composition, includes (a) c-kit-ligand comprising a polypeptide, amino acid sequence which (i) is encoded by the nucleic acid sequence shown in Fig. 11A-11D (SEQ ID NO: 1), or (ii) the naturally occurring hematopoietic variant of the polypeptide (i); (b) a carrier suitable for use ex vivo, and (c) an effective amount of hematopoietic growth factor in an amount effective to increase the levels of peripheral blood cells ex vivo.

 

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