NOVEL PHYSIOLOGICALLY ACTIVE PROTEINACEOUS SUBSTANCE

Abstract: A novel physiologically active proteinaceous substance having a DNA synthesis-accelerating effect, and a process for preparing this substance by culturing human myelogenous leukemia cell strain. Prospective applications of this sub­ stance include an immunoactivating agent, anemia-treating agent, wound-treating agent, and an additive to a serum-free medium for macrophage cells, fibroblasts, lymphoblasts, etc. as their growth factor.

Description: Specification Novel Physiologically Active Proteinaceous Substance Technical Field The present invention relates. to novel physiologically active proteinaceous substances having an activity of promoting DNA synthesis, and to a process for producing the said substances by culturing human myelogenic leukemia cells. Background Art Heretofore, the following examples have been known as substances having an activity of promoting DNA synthesis. (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 factorj 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-I (Insulin like growth factor) J. Endocr. 85, 267 (1980) (7) IGF-II (Insulin like growth factor) Diabetes 31, 2823 (1982) (8) MSA (Multiplication stimulating activity) Nature 272 (23), 776 (1978) These substances having an activity of promoting DNA synthesis are produced using media containing proteinaceous substances such as serum, etc., and thus it is difficult to identify the substances as those produced by cultured cells and to separate the substances from the medium components. Owing to these difficulties, these substances have not yet a practical significance. Disclosure of the Invention The present inventors have found that, when human myelogenic leukemia cells are cultured in a protein-free medium, the culture supernatant contains substances which remarkably promote uptake of 3H-thymidine into cells and promote DNA synthesis in an uptake test system using mouse fibroblast strain 3T3 and chick embryo fibroblast, and have established the present invention. The present invention is described in detail below. The present invention provides novel physiologically active proteinaceous substances having an activity of promoting DNA synthesis. The substances of the present invention can be classified into two groups, i.e., the one having a'molecular weight of 6,300 + 500 and the one having a molecular weight of 9,000 - 13,000, and can be further classified into the following species according to isoelectric points. Table 1 No. Molecular weight Isoelectric point (pI) 1 9,200 + 500 8.55 + 0.1 2 10,000 + 500 6.9 + 0.1 3 9,400 + 500 8.25 + 0.1 4 10,000 t 500 6.7 + 0.1 I 5 10,500 t 500 5.8 + 0.1 6 10,500 + 500 5.6 t 0.1 7 10,500 t 500 6.09 + 0.1 8 11,000 1 500 4.7 t 0.1 9 11,800 + 500 4.19 t 0.1 10 12,800 + 500 3.54 t 0.1 11 6,300 + 500 8.0 + 0.1 12 n 8.4 t 0.1 II 13 n 8.5 + 0.1 14 N 9.0 1 0.3 15 n 9.6 + 0.3 The substances of the present invention are not decomposed by deoxyribonuclease, ribonuclease and neuraminidase, and are inactivated by proteinase such as trypsin, chymotrypsin, etc. Thus, they can be regarded as proteinaceous or peptidic substances. The substances of the present invention have an activity of promoting uptake of 3H-thymidine into chick embryo fibroblasts and mouse fibroblast strain 3T3 and promoting DNA synthesis. Furthermore, the substances of the present invention also promote propagation of mouse fibroblast strain 3T3. Molecular weights and isoelectric points of said well known active substances are given below for comparison. Isoelectric point Substance Molecular weight (pI) PDGF 13,000 - 16,000 9.8 TGF 7,400 EGF 6,000 4.5 FGF 13,000 9.7 FSLA 29,000 4.7 16,500 6.1 - 6.3 IGF - I 7,649 8.3 IGF - II 7,500 6.5 MSA 40,000 20,000 11,000 7,000 It is evident that the substances of the present invention are novel substances because none of them have the same molecular weight or isoelectric point as these known substances and also they have a different origin. The substances of the present invention are formed and accumulated mainly in a culture supernatant by culturing human myelogenic leukemia cells in a medium, and thus can be recovered therefrom. Any of human myelogenic leukemia cells can be used so long as they can produce the substances of the present invention, and K562-Tl strain can be mentioned as a suitable strain. The K562-T1 strain consists of cells habituated to a protein-free medium, which are obtained by culturing K562 strain in Ham F10 medium initially containing 10% fetal calf serum while lowering the serum concentration successively for a habituation period of about one year. The K562 strain is a well known cell strain disclosed in Proc. Natl. Acad. Sci., USA., 76, 1293 (1979), Blood, 45, 321 (1975), GANN 73, 97 (1982), etc. and is generally available. As the medium, protein-free media such as Ham F10 medium and Ham F12 medium (products of Flow Laboratory, Inc.), Dulbecco MEM medium, MEM medium and RPMI-1640 medium (products of Nissui Seiyaku Co., Ltd.), etc., and their mixed media can be employed. Appropriate amounts of glutamine (0.5 - 5 mM/mi), antibiotics [penicillin (25 U/mL), streptomycin (25 pg/mi), etc.], sodium bicarbonate (0.01%), etc. may be added to the medium if necessary. For the culturing, various culturing bottles, Petri dishes, roller bottles, spinner flasks, jar fermenters, etc. can be employed. Culturing is usually carried out at a seed cell density of 5 x 104 - 1 x 106 cells/mQ and 30 - 400C for 2 - 4 hours whereby the substances of the present invention are formed mainly in a culture broth according to the respective cell density. For example, by culturing at the seed cell density of 1 x 105 cells/mQ and 370C for 2 days, 300 unit/ml active substance is formed in the culture broth. The substances of the present invention are recovered from the culture broth in the following manner. That is, the supernatant of the resulting culture broth is concentrated by freeze-drying, ultrafiltration, strongly acidic ion exchange resin, etc. Elution is carried out with a weakly basic buffer or an alkali solution of low concentration. Furthermore, low molecular substances of medium origin are removed by gel-filtration. As the gel filtering agent, Sephadex (made by Pharmacia Fine Chemicals, Inc.), Biogel (made by Bio-Rad Laboratories, Inc.), Control Pore Glass (made by Corning Glass Works, Inc.), Toyopearl (made by Toyo Soda Co., Ltd.), etc. can be employed. Since the substances of the present invention have a high molecular weight, many low molecular substances having an influence upon the biological activity assays can be removed by restricting the molecular weight-fractionating range to 1,000 - 5,000. By this operation, the active fractions can be fractionated substantially at the void volume position. The recovery of the active fractions is about 80%, and when only the measurement value at 280 nm is calculated and converted to the absorption at 280 nm of bovine albumin among the three wavelength measurements at 280 nm, 260 nm and 340 nm, the specific activity is increased about 200-fold. These active fractions are joined together and concentrated by freeze-drying. Then, the active fractions are fractionated according to molecular weights. The active fractions in an amount of 1/100 of the gel amount are passed through a column of biogel or Sephadex, and then elution is carried out with 0.05M phosphate buffer as an eluting solution at a flow rate of 0.1 in S.V. The active fractions are separated according to the respective molecular weights, and fractionated into three groups. The groups are designated as Fractions I, II and III in the order of molecular weight, I being the highest, and the ratio of I: III becomes 7:4:3. The entire activity recovery shows a substantial quantitative recovery in this step and the specific activity is increased 720-fold. For isoelectric point fractionation, these active fractions are desalted with strongly basic ion exchange resin such as Dowex 50W x 8 (made by Dow Chemical Co., Inc.), Amberlite IR-120 (made by Bio-Rad Laboratory, Inc.), SK102 (made by Mitsubishi Kasei Kogyo Co., Ltd.), SP-Sephadex C-25 (made by Pharmacia Fine Chemicals, Inc.), etc. After adsorption at pH 3 - 7, the column is washed with distilled water and eluted with 0 - 0.3N aqueous ammonia. The recovery in this step is 30 - 50%, and the activity ratio of the fractions is I:II:III = 7:2:3. Isoelectric point electrophoresis is carried out at 900V and 10 mA for 48 hours in a column with 100 mQ of LKB8100 (made by LKB, Sweden) at an ampholite concentration of 1.9 - 0.6258 and at a pH gradient of 3 - 10 while cooling to 40C. After the completion of electrophoresis, fractionation is carried out using a fraction collector, and pH and activity of the respective fractions are measured. In this step, 5 species of active fractions shown in the said group I can be obtained from Fraction I, and 6 species of active fractions shown in the said group II can be obtained from Fraction II, while no characteristic active species are obtained from Fraction III. The ampholites are removed from the respective fractions through a PD-10 column (G-25 column made by Pharmacia Fine Chemicals, Inc.). In this step, the recovery is 0.5 - 2% as the ultimate recovery, and the specific activity is increased 3,600 to 14,400-fold. Procedures for determining biological activities of the present invention are described below. 1. Evaluation of ability to promote DNA synthesis: A) Evaluation system using 3T3-L1 cells: Evaluation is carried out with a microplate with 24 holes (made by Nuncf Inc., Denmark) as a culturing vessel, a Dulbecco, et. al. - modified Eagle medium (DME medium, made by Nissui Seiyaku Co., Ltd.) containing glutamine (0.29 mg/mQ), sodium bicarbonate (0.1%), penicillin (15 units/mQ), streptomycin (15 vg/mQ) and heat-inactivated fetal calf serum (made by Flow Laboratory, Inc.) (medium containing 10% serum is hereinafter referred to as liquid I and that containing 0.4% serum as liquid II) and 3T3-Ll cells (ATCC92, available from Dai-Nippon Seiyaku Co., Ltd.). A cell suspension (2 x 104 cells/m±) is cultured in liquid I in air containing 5% carbon dioxide at 370C for 5 hours, and then the liquid I is exchanged with liquid II, followed by culturing for 24 hours. Then, 0.1 mQ of a sample is added, followed by culturing for 16 hours. 3H-thymidine is added and culturing is carried out for one hour. The amount of 3H-thymidine incorporated into DNA is measured with a liquid scintillation counter (made by Aroca, Inc.) B) Evaluation system using CEF (chick embryo fibroblast) cells: Evaluation is carried out with the same culturing vessel as used in A), a Ham F10 medium (made by Flow Laboratory, Inc.) containing glutamine (0.29 mg/mR), sodium bicarbonate (0.1%), penicillin (15 units/ml), streptomycin (15 Vg/mQ) and 0.4% heat-inactivated fetal calf serum (liquid III), an MEM medium containing glutamine (0.29 mg/mQ) and sodium bicarbonate (0.1%) (liquid IV) and 2nd generation CEF cells (obtained by incubating fertilized hen's eggs for 12 days to prepare first generation cells and then prepare the second generation after 3 days). A cell suspension (3.3x^105 cells/mQ) is cultured in liquid III in air containing 58 carbon dioxide at 370C for 72 hours. Theng the liquid III is exchanged with liquid IV, and 0.1 mi of a sample is added thereto, followed by the same operations as in A). C) Titer designation: Titer is calculated according to the procedure disclosed in Biochemistry 4 (2), 169 (1972), wherein an ability to incorporate 3H-thymidine into DNA shown by 100 ng/mQ insulin is designated as 1,000 units. 2. Procedure for determining an activity of promoting cell propagation with mouse 3T3 cells: Determination is carried out with a microplate with 24 holes (made by Nunc, Inc., Denmark), a Dulbecco, et. al. modified Eagle medium (DME medium, made by Nissui Seiyaku Co., Ltd.) containing glutamine (0.29 mg/mL), sodium bicarbonate (0.1%) , penicillin (15 units/mQ), streptomycin (15 vg/mQ) and heat-inactivated fetal calf serum (medium containing 10% serum is hereinafter referred to as liquid A and that containing no serum as liquid B), and 3T3-Ll cells. A cell suspension (3 x 104 cells/mQ) is cultured in liquid A in air containing 5% carbon dioxide at 370C for 4 hours. Then, the medium is exchanged with liquid B, and 0.1 mL of a sample is added thereto. After culturing for 48 hours, the number of cells is counted. The substances of the present invention having an action of promoting DNA synthesis on a broad range of cells have the following actions and various uses also. 1) They promote propagation of a- cell system having an ability to produce antibodies, and can be used as an immune activator. 2) They promote propagation of cells of erythrocyte system and thus can be applied to anemia. 3) They promote propagation of granulocytes and microphages, and an anti-cancer action can be expected owing to prevention of infection and immune-activating action. 4) They promote propagation of fibroblasts and thus can be applied as a curing agent for wounds. 5) They promote propagation of cells which produce tumor necrosis factor, etc. considered to have an anti-cancer action, and thus, an effect can be expected as a factor promoting propagation of cells of macrophage system or as a factor promoting propagation of fibroblasts, cells of lymphoblast system, etc. which produce interferon, by addition to a medium free from serum. These functions and effects are shown in Examples 2 and 3. Brief Description of the Drawings Fig. 1 shows an elution pattern of the substance of the present invention in Example 1 (1), wherein the activity refers to an activity of promoting the uptake of 3H-thymidine (the same shall apply hereinafter). Fig. 2 shows an elution pattern of the substance of the present invention in Example 1 (2). Fig. 3 shows an isoelectric point electrophoresis diagram of I Fig. 4 shows an isoelectric point electrophoresis diagram of II. Fig. 5 shows an'elution pattern of the substance of the present invention in relation to the molecular weight markers. Fig. 6 shows a relationship between the molecular weight and the eluting position. Fig. 7 is an elution pattern of the substance of the present invention in relation to the molecular weight markers. Best Mode for Carrying Out the Invention Example 1 (1) Myelogenic leukemia cell K562-T1 strain was subjected to stationary culture as seed cells at 370C for 4 days, and a cell supernatant was recovered. As the medium, Ham F10 medium (protein-free medium) containing glutamine (4 mM), penicillin (25 U/mA), streptomycin (25 pg/mQ), and sodium bicarbonate (0.01%) was used. The recovered cell supernatant (3,000 mQ) was subjected to centrifugation (6,000 rpm for 10 minutes), and the supernatant (3,000 m±) was freeze-dried. Dried powder (about 30g) was dissolved in distilled water (43 mi) to make the concentration 70 times that of the cell supernatant. The solution was subjected to gel filtration with Biogel P-6 (made by Bio-Rad Laboratory, Inc.). The amount of the sample was 1/20 (W/V) of the gel amount, and elution was carried out with 0.05M phosphate buffer (pH 7.0) at a flow rate of 0.1 in S.V. The eluate was recovered in 10 mQ fractions. Elution pattern is shown in Fig. 1. Low molecular substances were substantially completely removed, and an activity of promoting the uptake of 3H-thymidine appeared around the void volume (V0). The active fractions had also an effect of promoting the propagation of mouse 3T3-L1 cells, and thereby the number of cells propagated became 2.3 times as many as that in the case where no sample was added. The recovery was 80%, and the specific activity was increased 210-fold, when the absorption at 280 nm was used to obtain the protein content in terms of bovine albumin amount among three wavelength absorbances at 260 nm, 280 nm and 340 nm, using Shimadzu Spectrophotometer Us240. (2) Then, 60 m± of the said active fractions was collected, freeze-dried, and further fractionated likewise with Biogel P-60 (made by Bio-Rad Laboratory, Inc.). That is, 140 mg of the sample was dissolved in 4 mQ of distilled water, and the solution was passed through a 100-mQ column packed with Biogel P-60. Elution was carried out with 0.05M phosphate buffer (pH 7.0) at a flow rate of 0.1 in S.V. The eluate was recovered in 3 mb fractions. Elution pattern is shown in Fig. 2. Active fractions were separated into three groups, and can be separated from high molecular impurities around VO. The three active groups were substantially quantitatively eluted together, and the specific activity was increased 720-fold. The groups were designated as I, II and III, respectively, in the order of molecular weight, I being the highest, and the activity ratio of these active fractions was 7:4:3, while their specific activities were substantially equal to one another. (3) The active fractions I, II and III, (24 mi, 12 mi and 12 mL, respectively) were passed through a column packed with 1 mQ of a strongly acidic ion exchange resin, Dowex 50W x 8 (Na+ form, made by Dow Chemical Co.), and the column was washed with 15 mQ of distilled water. Elution was carried out with 25 mQ of distilled water and 25 mQ of a 3N aqueous ammonia according to the linear concentration gradient method. By this operation, salts were substantially completely removed. After the elution, the active fractions were freed from ammonia by freeze-drying. I II III Recovery 20% 9% 14% Specific activity 3,600 14,400 5,000 (4) Isoelectric points of the above fractions were determined using an 8100-1 column (made by LKB) with 1.9 - 0.625% of ampholite at pE 3 - 10. As an anolyte at the bottom of 110 mi-column was employed 1.0M phosphoric acid, and as a catholyte at the top was employed 1% ethylenediamine, and a concentration gradient of 50% - 5% was made by a gradient mixer. Electrophoresis was conducted at 900 volts and 40C for 48 hours, and after the separation, the sample was fractionated into a fraction collector in 1 mQ portions to measure pH. Biological activity was measured after separation of ampholite using PD-10 column (G-25 column made by Pharmacia Fine Chemicals, Inc.). Isoelectric point electrophoresis diagrams of I and II are shown in Figs. 3 and 4, respectively. The result on I showed pI values of 8.55, 6.9, 8.25, 6.7, 5.8, 5.6, 6.09, 4.7, 4.19 and 3.54, and the result on II showed pI values of 8.0, 8.4, 8.5, 9.0 and 9.6. Thus, it seems that I and II are mixtures of physiologically active proteinaceous substances having similar molecular weights and different amino acid compositions. No characteristic peak was observed in the case of III. (5) The active fractions obtained in (2) above were freeze-dried, and then dissolved in 0.05M phosphate buffer (pH 7.0) in a concentration of 15,000 U/mi. The solution was passed through a column of 1.5 cm inner diameter packed with 100 mt of Sephadex G-50 (made by Pharmacia Fine Chemicals, Inc.). Elution was carried out with 0.05M phosphate buffer (pH 7.0) at a flow rate of 0.05 in S.V. To obtain VO values, 3 mg/mi y-globulin (molecular weight: 160,000), 3 mg/mQ cytochrome C (molecular weight: 12,500, 3 mg/mQ insulin (molecular weight: 5,700) and 3 mg/mQ bacitracin (molecular weight: 1,450) were used as molecular weight markers. Since insulin has a low solubility under the neutral condition, it was eluted with 0.1N acetic acid. The eluting position of each marker was determined from the absorbancy at 280 nm. The results are shown in Fig. 5. To determine the molecular weights from the eluting positions, a relationship between the molecular weights and the eluting positions is shown in Fig. 6. These markers are positioned on a straight line on a semilogarithmic graph. Active fraction I showed molecular weights of 9,000 - 13,000, II 6,300 + 500, and III 2,400 + 500. It is seen from the foregoing results that the active fractions contain at least 15 species of substances shown in Table 1. (6) To determine the respective molecular weights, active fraction I obtained in (5) above was subjected to gel filtration by high pressure liquid chromatography. The high pressure liquid chromatography was carried out using BECKMAN HPLC 342 (made by Beckman, Inc.), and as the gel filtration column, TSK-GPC column G-3,000SW (7.5 x 600 cm) (made by Toyo Soda Kogyo Co., Ltd.). As the solvent, a mixture of 0.15M sodium chloride and 0.02M sodium acetate in the ratio of 1:1 was used at pH 6.0 as the molecular weight markers, 0.1 mg/0.2 mt cytochrome C (molecular weight: 12,500), 0.1 mg/0.2 mt trypsin inhibitor (molecular weight: 21,500), and 0.1 mg/0.2 mt aprotinin (molecular weight: 6,500) were used. The markers were positioned on a straight line on a logarithmic graph from their eluting positions. The components of the fraction were respectively subjected to gel filtration, and it was found that those appearing on the side of higher molecular weight by Sephadex G-50 had the following molecular weights. No. Molecular weight Isoelectric point (pI) 1 9,200 + 500 8.55 t 0.1 2 10,000 + 500 6.9 + 0.1 3 9,400 t 500 8.25 t 0.1 4 10,000 t 500 6.7 + 0.1 I 5 10,500 + 500 5.8 + 0.1 6 10,500 + 500 5.6 t 0.1 7 10,500 t 500 6.09 + 0.1 8 11,000 + 500 4.7 t 0.1 9 11,800 + 500 4.19 t 0.1 10 12,800 + 500 3.54 + 0.1 (7) Molecular weight of the components of the active fraction II obtained in (5) above having pI values of 8.4, 9.0 and 9.6 were determined by high pressure liquid chromatography. Analysis was made using HSG-30W column for gel filtration and Shimadzu HPLC, LC-4A. Detection was made with UV at 280 nm, and 20 iit of the sample was injected. As the eluting solution, a buffer of 0.1M KH2P04, 0,1M Na2HP04 and 0.1M Na2S04 (pH 6.8) was passed at a rate of 0.7 mQ/min. As the molecular weight markers, 0.2 mg/mi each of albumin (molecular weight: 69,000, ovalbumin (molecular weight: 45,000), cytochrome C (molecular weight: 12,500) and insulin (melecular weight: 5,700) were used, and the calibration curve shown in Fig. 7 was obtained. It was found that each component had a molecular weight of 6,000. Example 2 Action of promoting DNA synthesis on human lymphoblast cells: Active fraction I obtained in Example 1 was examined on the activity of promoting DNA synthesis of Namalva strain of the lymphoblast cells derived from Burkitts lymphoma. The Namalva strain was cultured at 370C in a medium prepared by adding glutamine (4 mM), streptomycin (25 pg/mQ), penicillin (25 U/mQ), HEPES (10 mM), sodium bicarbonate (0.01%) and fetal calf serum (108) to RPMI-1640 medium made by Nissui Seiyaku Co., Ltd. The cells were collected by centrifugation, and suspended sin the said medium freed from the fetal calf serum at a concentration of 5 x 105 cells/mt. The suspension was poured in portions into a multidish plate with 24 holes and cultured at 370C. After 3 days, the medium was exchanged with a fresh medium, followed by culturing at 370C for 2 days. Then, the medium was exchanged again with a fresh medium, and samples (dilutions of active fraction I) and comparative samples (FCS and insulin) were added thereto. After culturing at 370C for 6 hours, tritium thymidine was added thereto to 0.5 pCi/well, and the activities of its uptake were measured according to the ordinary procedure. The measurement values are averages of two runs of each test. Sample dilution Comparative sample 3H-TdR FCS Insulin x 2 x 4 x8 x 16 0.5% 100 ng/mi 0,5% 100 ng/ml Uptake cpm 365 305 153 260 338 431 -194 Example 3 Activity of promoting DNA synthesis on human leukemia cells: Active fraction I obtained in Example 1 was examined on the ability to promote DNA synthesis of various leukemia cells. K562 strain is an unmature leukemia cell strain which is considered to be differentiable into an erythrocytic system and has been disclosed in Blood 45, 321 (1975) as a human myelogenic leukemia cell strain. ML-l strain has been established as an acute myelogenic leukemia (myeloblast) cell strain in Cancer Res. 42 5152-5158 (1982) and is considered to be differentiable into macrophage monocytes. U-937 strain is a human hystocytic leukemia cell strain registered as ATCC CRL1593 and is a monocyte-like cell strain THP-1 strain is a strain disclosed in Int. J. Cancer 26 171-176 (1982) as human acute monocytic leukemia cell and is considered to be differentiable into macrophage. An activity of promoting DNA synthesis on these cell strains which are considered to be in a series of differentiating steps was measured in the following manner. Each of these cell strains was cultured at 370C in a medium prepared by adding glutamine (4 mM), streptomycin (25 pg/mQ), penicillin (25 U/mt), HEPES (10 mM) and sodium bicarbonate (0.01%), and further fetal calf serum (5%) to RPMI-1640 medium made by Nissui Seiyaku Co., Ltd. The cells were suspended in the said serum free medium. The suspension was poured in portions into a microtiter plate with 96 holes to 1 x 104 cells/well. The suspensions of ML-l and U-937 were cultured for 24 hours and those of K562 and THP-1 were cultured for 3 days. Then, 10 ut each of samples of various concentrations were added to the titer plates and culturing was conducted for 16 - 20 hours. Then, 0.1 uCi of tritium thymidine was added to conduct pulse labelling for 8 hours. After the labelling, the cells were collected onto a filter paper with an autoharvester, and the filter was washed with 3 mt of PBS (phosphate buffer solution containing 40g of NaCl, lg of KC1, 5.75g of Na2HP04 and lg of KH2PO4 in 5l of water) and further with 5% trichloroacetic acid and acetone, and dried under an infrared lamp. Then, the thymidine uptake was measured according to the conventional procedure. The measurement values shown below are averages of 3 runs of each test. Sample dilution K562 U-937 ML-1 THP-1 cpm cpm cpm cpm xl - - 607 947 x 2 948 799 470 2307 x 4 1086 679 475 1333 x 8 975 406 268 764 x 16 881 377 359 x 32 809 229 429 FCS 10% 449 535 680 1606 - 283 242 327 831

Claims: Claims 1. Novel physiologically active proteinaceous substances exhibiting an activity of promoting DNA syntho -s which are produced by a myelogenic leukemia cell strain. 2. A process for producing novel physiologically active proteinaceous substances, characterized by culturing a myelogenic leukemia cell strain in a protein-free medium, accumulating a physiologically active protein exhibiting an activity of promoting DNA synthesis in the culture broth, and recovering the protein from the culture broth.

Inventors: FUJIWARA, KAZUMI, FUJIYOSHI, NOBUO, OGATA, TOMOKO, OKABE, TETSURO, SATO, SEIJI
Applicants: KYOWA HAKKO KOGYO CO., LTD., OKABE, TETSURO
Application year: 1984
Main classification: C07K15
IPC codes: C07K14, C12N5, C12P21