JPH0262560B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a novel physiologically active proteinaceous substance that has cell proliferation-promoting effects and DNA synthesis-promoting effects. The substance of the present invention is expected to be used as an immunostimulant and an anticancer agent. Prior Art Conventionally, the following examples are known as substances having DNA synthesis-promoting effects and cell proliferation-promoting effects. (1) PDGF (Platelet-derived growth factor)
Proc. Natl. Acad. Sci., 76 , 1809 (1979) (2) TGF (Transforming growth factor) J.
Biol.Chem. 257 , 5220 (1982) (3) EGF (Epidermal growth factor) Anal.
Biochem. 111 , 195 (1981) (4) FGF (Fibroblast growth factor) Ann.Rev.
Biochem. 45, 531 (1976) (5) FSLA (Fibroblast Somatomedin like
activity) J. Cellular Physiology 107 , 317 (1981) (6) IGF− (Insulin like growth factor) J.
Endocr. 85, 267 (1980) (7) IGF− (Insulin like growth factor)
Diabetes 31, 2823 (1982) (8) MSA (Multiplication stimulating activity)
Nature 272 (23), 776 (1978) Among these, the primary structures of substances No. 1 to 4, 6 and 7 have been clarified, but they clearly differ from the substance of the present invention in their N-terminal structure. Other substances are difficult to separate from medium components and have no practical value. JP-A-59-181293 discloses a protein substance with DNA synthesis promoting activity produced by myeloid leukemia cells, which is clearly different from the substance of the present invention in terms of molecular weight and other properties. Problems to be Solved by the Invention and Means for Solving the Problems In order to obtain useful immunostimulants and anticancer agents, the present inventors searched for substances that have the effect of promoting DNA synthesis and cell proliferation in animal cells. As a result, we found that when myeloid leukemia cells are cultured in a protein-free medium, a new substance that significantly promotes the growth of Burkitt's lymphoma-derived lymphoblastoid cells accumulates in the culture supernatant. This substance is a proteinaceous substance that promotes DNA synthesis and cell proliferation in animal cells, and based on its remarkable activity, it is expected to be used as an immunostimulant and an anticancer agent. Components of the Invention The present invention provides a novel proteinaceous substance that has the effect of promoting DNA synthesis and cell proliferation in animal cells. The substance of the present invention has the following physical and chemical properties. (1) Molecular weight: 20000±2000 Molecular weight is SDS (sodium dodecyl sulfate) −
Polyacrylamide gel electrophoresis [SDS-
PAGE method, New Experimental Chemistry Course (20) Biochemistry ()
p118, edited by Memoto Chemical Society, Maruzensha Publishing]. As a result of analysis with 3% concentration gel and 16% acrylamide concentration on separating gel, the flow sample volume was 20 μl.
showed a single band. For staining, silver staining reagent "Daiichi" manufactured by Daiichi Chemical Co., Ltd. was used. As molecular weight markers, lysozyme (MW14400),
Soybean trypsin inhibitor (MW21500), carbonic anhydrase (MW31000), ovalbumin (MW45000), bovine serum albumin (MW66200) and phosphorylase B
(MW92500) was used. All molecular weight markers
Manufactured by BIO-RAD Lab. (2) N-terminal protein primary structure Met-Gln-Ile-Phe-Val-Lys-Thr-Leu
−Thr−Gly−Lys−Thr−Ile−Thr−Leu−Glu
-Val-Glu-Pro-X-Asp-X-ILe-X-
Asn-Val-X-Ala-X-Ile- (X indicates an unidentified amino acid.) The amino acid sequence was determined using an Applied Biosystems 470A sequencer and Spectra Physics.
Determination was made in combination with high performance liquid chromatography (manufactured by Physics). (3) Thermal stability: Stable for processing at PH7.3, 50℃, 30 minutes. The substance of the present invention (lyophilized powder) was added to PBS (-) (NaCl 8
g/, KCl 0.2g/, Na ₂ HPO ₄ 1.15g/,
KH ₂ PO ₄ 0.2g/) in water solution at 50℃
After heating for 30 minutes, cell proliferation promoting activity was measured using lymphoblastoid Namalva strain by the following method. (4) PH stability: Stable for 24 hours at 4℃ and PH 2-3. The culture supernatant of the K562-T1 strain was concentrated 200 times using a holographic fiber with a molecular weight of 3000, and this was dialyzed using 0.1% acetic acid at 4°C for 24 hours to determine the cell proliferation promoting activity of the dialysate. It was measured using the Namalva strain by the following method. (5) Isoelectric point: 6.5±0.5 Isoelectric focusing method is performed using 110ml of LKB8100 (LKB
Using an ampholyte ampholyte column (Sweden), the concentration of ampholyte ampholyte was 1.9 to 0.625%, and the pH was 3 to 10.
Use the slope of Cooled to 4℃, 900V, 10mA
After 48 hours of electrophoresis, fractionate using a fraction collector and measure the PH and activity of each fraction. (6) DNA synthesis promoting activity and cell proliferation promoting activity
DNA synthesis promotion activity measurement method Test cells 1 to 5 x ¹⁰⁵ cells/ml were added to RPMI-1640.
25 ml of medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing glutamine (4mM), streptomycin (25μg/ml), penicillin (25U/ml), Hepes (10mM), baking soda (0.01%), and calf serum (10%). The cells were cultured at 37°C for 2 days. The cells were collected by centrifuging the culture at 800 x g for 5 minutes, suspended in the above medium minus calf serum to a concentration of 5 x 10 ⁵ cells/ml, and divided into 24-well multi-dish plates. After culturing at 37℃ for 24 hours, replace with fresh medium.
Culture was performed at 37°C for 16 hours. The medium was again replaced with fresh medium, and 0.1 ml of the sample and 0.9 ml of the medium were added. 37℃, 6
After culturing for an hour, tritiated thymidine was added at a concentration of 0.5 μCi/well, and its uptake activity was measured using a liquid scintillation counter. The measured value is
The average value of two or more experiments is shown. Cell proliferation promoting activity Test cell line 5-10×10 ⁵ cells/ml with RPMI-
1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing glutamine (4mM), streptomycin (25μg/ml), penicillin (25U/ml), Hepes (10mM), baking soda (0.01%), and calf serum (10%).
It was cultured in 75 ml at 37°C for 48 hours. culture at 800 x g;
Collect the cells by centrifuging for 5 minutes, wash with the above medium minus serum, dispense into a 24-well multi-dish plate, and add 0.9 ml of serum-free medium.
0.1 ml of sample solution was added. After culturing for 3 days at 37°C in a CO ₂ incubator, the number of cells was measured using a hemocytometer. For adhesion-dependent cells, immediately before measurement,
Measurement was performed after suspending the cells by adding 0.1% trypsin. The activity is 100 for the sample without additives.
The value is calculated by dividing the number of cells when the sample is added by the number of cells when the sample is not added. The DNA synthesis promoting activity and cell proliferation promoting activity for various cells of the substance of the present invention obtained by the above measurement method were as shown in Table 1. All cells in Table 1 except those marked with * are human-derived cells. [Table] Indicates genderlessness.
As mentioned above, the substance of the present invention has DNA synthesis promoting activity and cell growth promoting activity against various animal cells, and is expected to have the following uses. (1) Since it promotes the proliferation of B-cell cells capable of producing antibodies, it can be used as an immunostimulant. (2) It promotes the proliferation of Erythro leukemia cells (K562), so it can be applied to anemia. (3) Since it promotes the proliferation of T-cell cells, it can improve cellular immune function and can be used as an anticancer agent. (4) It can be used as an anticancer agent because it promotes the proliferation of granulocytes (K562) and macrophage cells (monocytes), and as a therapeutic agent for wounds because it increases DNA synthesis in epithelial cells. (5) Since it promotes the proliferation of B-cell and Myeloma cells, it can be applied to in vitro antibody production. Considering the various properties of the substance of the present invention, ubiquitin fractionated from calf thymus can be cited as a substance similar to the substance of the present invention. The 30th amino acid sequence from the N-terminus of the substance of the present invention shows almost the same sequence as ubiquitin. However, since ubiquitin has a molecular weight of approximately 8,500 and does not exhibit a proliferation-promoting effect on Namalva cells, it is clear that the substance of the present invention is a substance different from ubiquitin. Substances with piston proteins attached to ubiquitin and precursors with linked ubiquitin molecules are known [Nature 225, 423, 1975; Journal of
Biological Chemistry (JBC), 250,
7182, 1975: Nature 31 2 , 663,
1984]. These substances also differ in molecular weight etc.
Furthermore, the biological activities of these substances have not been clarified. Recently, there is a report that a ubiquitin precursor gene has been discovered [JBC 260, 7609, 1985].
There is no mention of producing proteins using this.
Furthermore, since the amino acid sequence encoded by this gene contains many basic amino acids, it is thought to be degraded by proteolytic enzymes in vivo, and thus does not exist as a protein. The substance of the present invention can be produced as follows. The substance of the present invention can be produced by culturing human myeloid leukemia cells in a medium, allowing them to accumulate in the culture, and collecting them from the culture. As the human myeloid leukemia cells used in the present invention, any cells that can produce the substance of the present invention can be used. As a suitable example, myeloid leukemia cell line K562-T1 can be used. The K562-T1 strain is a protein-free medium-acclimated cell obtained by sequentially lowering the serum concentration of the K562 strain from Ham's F10 medium containing 10% fetal calf serum and undergoing an acclimatization period of about 1 year. The K562 stock is owned by the Proceedings of the National Academy of Sciences.
Of Science (Proc.Natl.Acad.Sci.),
USA., 76 , 1293 (1979), Blood,
45, 321 (1975), GANN, 73 , 97 (1982)
This is a commonly available cell line among the known cell lines described in, etc. Media include Ham's F10 medium, Ham's F12 medium (manufactured by Flow Lab), Dulbecco's MEM medium,
Protein-free media such as MEM medium and RPMI-1640 medium (all manufactured by Nissui Pharmaceutical Co., Ltd.) and mixed media thereof are used. Glutamine may be added to the medium if necessary.
0.5-5mM, antibiotics [penicillin (25U/ml),
An appropriate amount of streptomycin (25 μg/ml), baking soda (0.01%), etc. may be added. For culturing, various culture bottles, shears, roller bottles, spinner flasks, jar fermenters, etc. can be used. The culture medium usually has a seed cell density of 5 x 10 ⁴ to 1 x 10 ⁶ cells/ml, with a seed cell density of 30 to 40
℃ for 2 to 4 days, the substance of the present invention is mainly produced in the culture solution depending on each cell density. For example, 1
At a seed cell density of ×10 ⁵ cells/ml and incubation for 2 days at 37° C., 300 units/ml of active substance are produced in the culture medium. The substance of the present invention is collected from the culture as follows. That is, the obtained culture supernatant is concentrated using freeze-drying, ultrafiltration, strongly acidic ion exchange resin, or the like. For elution, use a weakly basic buffer or a low concentration alkaline solution. Since the concentrated solution contains many low-molecular salts and impurities, it is dialyzed with 1% acetic acid. This removes much of the impure protein as a precipitate. Acetic acid is removed by freeze-drying, and the product is further concentrated to obtain a crude product. The crude product is passed through a cation exchange resin. The active substance has only a weak interaction with the cation exchange resin and is not adsorbed but eluted. The active fractions are collected, concentrated by freeze-drying, etc., and then low-molecular substances derived from the medium are removed by gel filtration. Gel filtration agents include Cephadex (Pharmacia,
(manufactured by Huain Chemical Co., Ltd.), biogels (manufactured by Biorad Co., Ltd.), control pore glass (manufactured by Corning Glass Works Co., Ltd.), Toyo Pearl (manufactured by Toyo Soda Co., Ltd.), etc. are used. When Bio-Gel P-60 or the like is used, the active fraction is located between the void volume (Vo) and low molecular weight substances such as salts. The concentrate obtained by the above operation can be purified as a single component by further performing high performance liquid chromatography. Example 1 8×10 ⁵ myeloid leukemia cell line K562-T1
The cells/ml were placed in 20 large spinner flasks containing 8 of the following medium and cultured at 37°C for 3 days. The culture medium was Ham-F10 medium (manufactured by Flow Lab) and MEM of Darbetsuko et al.
Pyruvic acid (5mM) and selenite (1.25mM) were added to a medium (manufactured by Nissui Pharmaceutical Co., Ltd.) mixed at a ratio of 3:1.
×10 ^-7 M), galactose (1 mg/ml), glutamine (4 mM), penicillin (25 U/ml), streptomycin (25 μg/ml) and baking soda (0.01%)
A protein-free medium supplemented with was used. Culture supernatant 120
was concentrated to about 100 ml using a hollow fiber (molecular weight KATTO-3000, manufactured by Amicon). Concentrate approx.
A 100 mm sample was dialyzed against 1% acetic acid at 4°C for 24 hours.
Centrifuge the resulting precipitate (12000 rpm, 10 minutes)
The supernatant was lyophilized to remove acetic acid to obtain a powder. This was dissolved in 120 ml of distilled water. The solution obtained above was passed through a 160 ml QAE-Sephadex-A50 (manufactured by Pharmacia Huain Chemical Co.) column in two portions of 60 ml each.
Elute with 150 ml of PBS(-), then add 0.1% acetic acid 190
ml and active fraction eluted with PBS(-) 300
Got ml. The activity recovery rate was approximately 8.3%, and the activity increased 1.9 times. 300 ml of this active fraction was freeze-dried, dissolved in 20 ml of distilled water, and passed through a column containing 400 ml of Bio-Gel P-60 in two portions at SV 5.0. PBS(-)
The eluate was fractionated into 6 ml portions to obtain 60 ml of the 41st to 45th fractions. The recovery rate of activity was 1.7%;
Specific activity increased 13.3 times. This active fraction was analyzed using FPLC (Fast) manufactured by Pharmacia Huain Chemical Co., Ltd.
protein liquid chromatography) Mono S 5
It was passed through a column containing ml. Washed with PBS(-) and eluted with 0-0.5M saline. 1ml of eluate
The activity was found in the 53rd fraction. The recovery rate of activity was 0.2%, and the specific activity increased 3600 times. This active substance is called LGF-1. The results of the above purification process are shown in Table 2. [Table] Example 2 Purification step Bio-Gel obtained in Example 1
Using the P-60 active fraction, Namalva cells (B-cell
), K562−T1 (Erythroleukemia, non T
The proliferation-promoting activity for each cell (non B type) and CCRF-CEM (T-cell type) was measured as described above.
The results are shown in Table 3. [Table] Example 3 Purification step Bio-Gel P obtained in Example 1
−60 active fraction was used for KB cells and HeLa.
Cellular DNA synthesis promoting activity was measured as described above. Table 4 shows the ³ H-thymidine incorporation rate when the active fraction was added, with the amount of ³ H-thymidine uptake taken as 1 when no active fraction was added. [Table] Effects of the invention
Provided is a novel proteinaceous substance that has DNA synthesis-promoting effects and cell proliferation-promoting effects.

Claims (1)

[Claims] 1. A novel proteinaceous substance having the following physicochemical and biological properties. (1) Molecular weight: 20000±2000 (SDS-PAGE method) (2) N-terminal protein primary structure Met-Gln-Ile-Phe-Val-Lys-Thr-
Leu−Thr−Gly−Lys−Thr−Ile−Thr−Leu
-Glu-Val-Glu-Pro-X-Asp-X-Ile-
X-Asn-Val-X-Ala-X-Ile- (X represents an unidentified amino acid.) (3) Thermal stability: Stable when treated at PH7.3, 50°C for 30 minutes. (4) PH stability: 4°C, PH 2-3, stable for 24 hours of treatment (5) Isoelectric point: 6.5±0.5 (6) Has a proliferation-promoting effect on the following cells. Human B lymphoid cells Human T lymphoid cells Human Myeloma cells Human Monocyte cells Human non-T non-B cells (7) It has a DNA synthesis promoting effect on the following cells. Human B lymphoid cells Human T lymphoid cells Human Myeloma cells Human Monocyte cells Human non-T non-B cells Human epithelial cells