JPH0532025B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a human transverse strangulation cell carcinoma cell mutant strain KYM-A that can be cultured under suspension under agitation. More specifically, the present invention relates to a human transverse strangulation cell carcinoma cell mutant KYM-A that produces a significant amount of the plasminogen activator KYM by suspension agitation culture. In recent years, diseases caused by blood clots have been highlighted as an important problem, and streptokinase, urokinase, etc. have been used to treat them. but,
All of them are known to not only dissolve thrombosis, which is the objective, but also decompose coagulation factors and the like in the circulating blood, resulting in a bleeding tendency. The cause of this is
Streptokinase, urokinase, and the like have poor affinity with the thrombus to be dissolved, and are thought to degrade blood coagulation factors and the like in the circulating blood.
Therefore, there has been a need to develop a thrombolytic agent that has a high affinity for blood clots and has fewer side effects such as bleeding tendency. Therefore, research is being conducted on plasminogen activators (hereinafter referred to as PA), which are said to dissolve blood clots and have few side effects, and presentations have also been made on this topic. Regarding human-derived PA, detailed studies have been conducted on enzymes obtained from endothelial cells and normal tissues such as the uterus, as well as tumor tissues and cultured cells thereof. (References include Cancer Research 40 933-938 (1980) by Wilson et al. and Journal of Biological Chemistry 256 7035-7041 (1981) by Ritsken and Collan). It has been known that normal tissue cells such as human endothelial cells and human uterine cells, as well as tumor cells such as human melanoma and breast cancer cells, produce PA. However, normal tissue cells eventually die, so they cannot be used for industrial production. In addition, the conventional culture of tumor cells such as human melanoma and breast cancer cells is limited to adhesion culture in bottles, etc., and the productivity of PA is extremely low, and it has not been possible to mass-produce it industrially. Therefore, the present inventors conducted research in search of tumor cells that can be cultured in floating agitation, and as a result, they discovered and isolated a mutant strain that can grow in suspension from a culture of KYM-I, a cell isolated from human lateral strangular myoma. He succeeded in doing so. This mutant strain is mutant strain KYM-A
It was named. The mutant strain KYM-A of the present invention is a mutant strain that was discovered through repeated separation and selective culture from a culture of KYM-I cells isolated from human lateral strangulation myoma. It is extremely distinctive in that it produces a significant amount of. This mutant strain KYM−
A can be subcultured by floating agitation culture, but the deposit at the Institute of Fine Technology was not accepted. The properties of mutant strain KYM-A are as follows. 1. Individual cells exhibit a highly refractive spherical shape. 2 Cells may exist singly as floating cells, but may also form chain-like or spherical aggregates. The number of cells included in the aggregate is approximately 2~
There are 100 pieces. 3 When cultured in a plastic shed or agitated culture in a tank,
This mutant strain can grow mostly as floating cells. 4. This mutant strain can be subcultured by floating agitation culture. 5. This mutant strain transforms into adherent cells by conditioned medium, etc. The altered cells exhibit epithelial cell-like morphology. The acclimatization medium is a culture supernatant of cells other than this mutant strain, which is obtained by culturing appropriate cells in an appropriate basal medium for 1 to 100 hours, and removing cells and their fragments from the culture medium. It is. As cells other than this mutant strain, for example, human hepatoma cells (HuH6-cl5 strain and HuH7 strain) (Nakamura et al., Cancer Research, Vol. 42 3858-3863 1982) can be used. In order to make this mutant strain adhesive, in addition to using the above-mentioned conditioned medium, it is necessary to use a medium containing fibronectin, or to replace the adhesive surface of Shearley with collagen, gelatin, poly-L-lysine, or egg white lysozyme. Adhesive culture of this mutant strain becomes possible by treatment with basic proteins such as . 6 10 When ⁶ cells of this mutant strain are transplanted subcutaneously into nude mice or into the sac of ALS-treated hamsters, a tumor is formed. 7. Chromosomes When the number of chromosomes was determined according to the conventional method of cytogenetics, the most frequent chromosome numbers were 46 and 47, and the distribution showed a slight width around these numbers. Therefore, mutant strains
The chromosome number of KYM-A is similar to that of normal human diploid (chromosome number), regardless of the tumor origin.
46) was found to be relatively similar. 8 This mutant strain produces significant amounts of PA-KYM. Further research revealed that when this mutant strain KYM-A was cultured, it produced and accumulated a significant amount of PA in the culture solution. This PA is PA−
It was named KYM. It is generally believed that there are mainly two types of PA in humans. That is, one type is a urokinase type that has poor affinity for fibrin, and the remaining type is a so-called tissue plasminogen activator (TPA) type that has a high affinity for fibrin. Melanoma cells are well known as tumor cell lines that produce TPA-type PA. Melanoma cells have attracted attention because they mainly produce only TPA-type PA. on the other hand,
Breast cancer cells have been reported as an exception among other types of tumor cells that produce only TPA-type PA. Furthermore, previous research has suggested that lateral strangulation myoma cells produce only urokinase-type PA. Urokinase-type PA cannot be detected in the culture solution of mutant strain KYM-A. Therefore, PA-KYM is novel in its origin as it is produced from human lateral strangulation sarcoma cells, and its physicochemical properties indicate that it is a novel TPA type.
It is recognized as PA. The mutant strain KYM-A of the present invention may be cultured by either adhesive culture or floating agitation culture. Mutant strain KYM-A adheres to plastic shearley or glass shearley and proliferates, so it is possible to perform adhesive culture using roller bottles or microcarriers. However, floating agitation culture is suitable for industrial production. For production of PA-KYM, mutant strain KYM-
It is preferable to culture A cells in a serum-supplemented medium and change to a serum-free medium when cell growth enters the stationary phase. As the serum-added medium, RPMI (manufactured by Flow), MEM (manufactured by Flow), or a mixed medium of both, to which about 10% fetal bovine serum is added, is preferable. Also, as a serum-free medium, it can be used as a basic medium.
It is preferable to use RPMI, MEM, or a mixed medium of both, to which insulin, human transferrin, monoethanolamine, and selenite are added (this four-component supplemented medium is described below).
For the purpose of producing PA-KYM from mutant KYM-A cells (referred to as ITES supplemented medium), the cell line is cultured in suspension in a plastic shear array, and when an appropriate number of cells is reached, suspension agitation culture in a spinner flask is started. . The spinner flask preferably has a capacity of 100ml to 8000ml.
Further, the inoculated cell density is preferably 10 ⁴ to 10 ⁵ cells/ml, and the stationary phase is reached at a density of 5×10 ⁵ to 2×10 ⁶ cells/ml. The culture temperature is not particularly limited, but approximately 30 to 40°C is suitable, particularly preferably around 37°C. In addition, the gas phase is 100% air or 5~
Air containing 10% carbon dioxide, or 5-100
Air containing % oxygen is preferred. Batch culture may be used, but if the cells grow sufficiently, PA-KYM can be cultured for about 1 month after starting culture by continuously replacing the medium at intervals of 1 to 4 days.
A culture solution containing the following can be obtained. PA-KYM of mutant KYM-A cells is oxygen that activates plasminogen to plasmin, and since it belongs to proteins, any general method applied to protein purification can be applied. Generally, salting out, adsorption, affinity chromatography,
Applications of ion exchange chromatography, molecular sieves, etc., and appropriate combinations thereof can be considered. Further, these purification processes can be carried out either continuously or batchwise, and the method may be selected in consideration of the efficiency, operability, etc. of purification. Next, the physicochemical properties of purified PA-KYM will be shown. a A product of the human transverse strangulation cell carcinoma cell mutant KYM-A. b. Molecular Weight The non-reduced substance measured by SDS-polyacrylamide gel electrophoresis has two closely spaced bands with molecular weights ranging from about 56,000 to 62,000. Furthermore, the reduced form of this substance measured by the same method has two bands at molecular weights of about 32,000 and about 36,000. c. Action and substrate specificity This substance is an enzyme protein that causes a fibrin dissolution reaction in the presence of plasminogen.
Because this reaction requires the presence of plasminogen, it belongs to a group of substances called typical plasminogen activators, and when compared to urokinase, it exhibits a much stronger binding ability to fibrin than urokinase. Furthermore, when the synthetic substrate S-2444 is used as a substrate, Km is 1.31×10 ^-3 M, and V _nax is
It is 0.046IU/min・ml. d Optimum PH The optimal PH is about 8-10 and the action curve is shown in FIG. e Stable PH Stable PH is approximately 5-11, and the % remaining activity is shown in Figure 2. f Range of suitable temperature for action The action temperature curve is shown in Figure 3 in the range of 30 to 45°C. g. Temperature resistance After heating for 90 minutes, there is almost no loss of activity up to 50°C, but at temperatures above 60°C, residual activity becomes below 60°C. The % remaining activity is shown in FIG. h Inhibition Residual activity was examined at 0.1 mM, 1 mM, and 10 mM of each inhibitor to obtain the results shown in Table 1.

[Table] Inhibition and activation were investigated by setting the activity of the sample without any drug as 100%. i Amino acid composition When this substance, which is an enzyme protein, was hydrolyzed with 6N hydrochloric acid and subjected to amino acid analysis, the results shown in Table 2 were obtained. Amino acid composition was expressed as a percentage of the total number of amino acid residues.

【table】

[Table] Nawanakatsuta.
j Ultraviolet absorption spectrum The ultraviolet absorption spectrum of an aqueous solution of this substance is as shown in FIG. k Solubility in solvents The solubility in water and saline solutions such as phosphate buffer is approximately 50 μg/ml, and when preparing solutions with higher concentrations, the presence of a solubility promoter, such as 1.6 M potassium thiocyanate, is required. .
It is insoluble in organic solvents such as ethanol and ether. l Properties of the substance The lyophilized specimen is a white powder. m Color reaction When PAS reaction was performed after SDS-polyacrylamide gel electrophoresis, a pink coloration characteristic of glycoproteins was observed, indicating that it was a glycoprotein. In addition, this substance is concanavalin A
-It was suggested that it is a glycoprotein because it showed affinity for agarose resin. n Isoelectric point When this substance was analyzed by chromatography in the presence of 8M urea, the isoelectric point of the main component was PH
7.5 to 8.0, and the isoelectric point of the subcomponents is PH7.0 to 8.0.
7.5, indicating that it is a mixture of weakly basic proteins. Next, examples of the present invention will be shown. Example 1 Cultivation For culturing mutant strain KYM-A, 2.0 g of sodium bicarbonate and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were added per medium.
1.192 g, RPMI-1640 medium (Flow Laboratories, USA) containing 60 mg of kanamycin sulfate and heat-inactivated fetal bovine serum (KC Biological, USA) to a final concentration of 10%, or medium 1
MEM medium (Flow Laboratory, USA) containing 2.0 g of sodium bicarbonate, 60 mg of kanamycin sulfate, and heat-inactivated fetal bovine serum to a final concentration of 10%, or the above two types of media in a 1:1 ratio. Use a mixture of KYM-A cells are inoculated into a plastic shed, and when approximately 100 ml of cell suspension is obtained as a result of proliferation, floating agitation culture using a spinner flask is started. 8 floating agitation culture is possible by gradually increasing the size of the spinner flask. The initial seeding density at each stage is 10 ⁴ to 10 ⁵ cells/ml, and grafting is performed when the density reaches 5×10 ⁵ to 2×10 ⁶ cells/ml. Example 2 Harvesting When the cell density reaches 5×10 ⁵ to 2×10 ⁶ cells/ml, the medium is changed to serum-free medium. The medium is 1
per sodium bicarbonate 2.0g N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid 1.192
g, kanamycin sulfate 60 mg, insulin 8.5 mg,
RPMI containing 1 mg of human transferrin (Sigma), 4.6 mg of ethanolamine, 13 μg of selenite, and 20 KIU/ml of aprotinin (Sigma).
1640 medium. Suspension agitation culture is carried out in this serum-free medium for 1 to 4 days, and this is repeated 4 to 10 times, and the culture solution is filtered to obtain cells. One solution is used for purification of PA-KYM.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the action curve of each PH in PA-KYM, Figure 2 is a diagram showing the residual activity of each PH, and Figure 3 is a diagram showing the residual activity of each PH.
The figure shows the action temperature curve, FIG. 4 shows the residual activity at each temperature, and FIG. 5 shows the ultraviolet absorption spectrum.

Claims (1)

[Scope of Claims] 1. Human transverse strangular myoma cell mutant KYM-A having the following properties. 1. Individual cells exhibit a highly refractive spherical shape. 2 Cells may exist singly as floating cells, but may also form chain-like or spherical aggregates. The number of cells included in the aggregate is approximately 2~
There are 100 pieces. 3. When cultured in a plastic shed or agitated culture in a tank, this mutant strain can mostly grow as floating cells. 4. This mutant strain can be subcultured by floating agitation culture. 5. This mutant strain transforms into adherent cells by conditioned medium, etc. The altered cells exhibit epithelial cell-like morphology. When 6 10 ⁶ of these mutant cells are transplanted subcutaneously into nude mice or into the cyst of ALS-treated hamsters, they form a tumor. 7. Chromosomes When the number of chromosomes was determined according to the conventional method of cytogenetics, the most frequent chromosome numbers were 46 and 47, and the distribution showed a slight width around these. That is, KYM-A
The chromosome number of the strain is relatively similar to that of a normal human diploid (46 chromosomes), despite the fact that the cells are derived from a tumor. 8 This mutant strain is a plasminogen activator
Produce a large amount of KYM.