Purification and Characterization of a Cell Growth Factor from a Human Leukemia Cell Line: Immunological Identity with Ferritin 1

We have succeeded in long-term cultivation of a human erythroleukemia cell line, K-562-T1 (T. Okabe, M. Fujisawa, and F. Takaku, Proc. Natl. Acad. Sci. USA, 81: 453-455, 1984). The cells grown in a protein-free chemically defined medium have been shown to produce cell growth factors (A. Mihara et al., In Vitro Ceil. Dev. Biol., 23: 317-322, 1987). In this study, we have purified a cell growth factor from the conditioned medium that stimulates the proliferation of human leukemia cells, HL-60. In the purified factor, two major protein bands of 24 kDa and 22 kDa were identified on a sodium dodecyl sulfate-polyacrylamide gel. The 22 kDa protein was stained with a monoclonal antibody to the light chain of ferritin. The growth-promoting activity of the purified factor was coprecipitated with a monoclonal antibody to the light chain or heavy chain of human ferritin. These results suggest that K-562-T1 cells produce a cell growth factor that is related to ferritin. I N T R O D U C T I O N The ability of cancer cells to produce and respond to their own growth factor (autocrine growth factor) has become a central concept which is emerging as a unifying theme in the search for the molecular and cellular basis of malignant transformation (1). Many of malignant cells have been reported to produce growth factors in cell culture (2). The major difficulty in showing the direct evidence that the cells produce growth factors is the contamination of the growth factors by calf serum, which had been sequestered on the cell membrane or incorporated into cytoplasm and released into the med ium used for purification of the growth factors. To exclude this troublesome possibility, we have established human cancer cell lines in protein-free chemically defined media (3, 4). The use of cells grown in a proteinfree culture system provides a great advantage for the demonstration and purification of growth factors produced by the cells. K-562-T1 cells, established from an erythroleukemia cell line, K-562, have been well propagated in a protein-free chemically defined med ium for 12 years. No protein supplements were used in the culture. It is therefore apparent that growth factors present in the condit ioned med ium of this cell line are produced by the cells themselves. The present study describes purification and characterization of a cell growth factor from the human erythroleukemia cells grown in a protein-free chemically defined medium. MATERIALS AND METHODS Preparation of a Serum-free Conditioned Medium A human leukemia cell line, K-562-T1 was established from an erythroleukemia cell line, K-562, in a protein-free chemically defined medium. The cells have been grown in a protein-free synthetic medium (Dulbecco's modified Eagle's medium: Ham's F-10 nutrient mixture, 3:1, supplemented with 5.6 mra galactose, 5 mM sodium pyruvate, 0.1 mr~ sodium selenite, 100 units/ml peniReceived 7/19/93; accepted 11/1/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by The Nomura Foundation. 2 To whom requests for reprints should be addressed. cillin G potassium, and 0.1 mg/ml streptomycin sulfate) at 37~ in a humidified 5% CO2 atmosphere. The cells have been maintained in plastic flasks (A/S Nunc, Roskilde, Denmark) with about 38 h of population-doubling time for these 12 years. The conditioned medium was harvested at a cell density of 5-10 • 105/ml and filtered through a GA 100 glass filter (Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The filtrate was stored at -20~ until use. Measurement of the Growth-promoting Activity Growth-promoting activity was determined by using human myeloblastic leukemia cells, HL-60 (passage 30-50, from the American Type Culture Collection), as indicator cells. HL-60 cells were maintained in enriched RPMI 1640 (RPMI 1640 supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM glucose, 0.05 mM 2-mercaptoethanol, and antibiotics) with 10% fetal calf serum (Salmond Smith Biolab Ltd., Auckland, New Zealand). For mitogenic assay, the cultured cells in exponential growth phase were washed twice in fresh enriched RPMI 1640 to remove fetal calf serum and were inoculated into 96-well flat-bottomed plates (Becton Dickinson and Co., Franklin Lakes, NJ) at the initial cell density of 1 • 104 cells/well in 0.1 ml of enriched RPMI 1640 without fetal calf serum. Sample (10/xl) was added to each well and the plates were incubated at 37~ in a humidified atmosphere of 5% COn in air. After 5 days, the cell numbers were counted by MTr 3 assay (5). MTr dye was reduced by mitochondrial dehydrogenases to a purple formazan. The direct proportionality between dye reduction and cell number extended over a wide range of cells from 1 • 103 to 1 X 105 cells/well. Briefly, 10/zl of 0.5% MTr (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in PBS (-) were added to each well and the plates were incubated at 37~ for 4 h. After the addition of 100 ~1 of 0.04 N HC1 in isopropyl alcohol to each well, the cells were vigorously mixed by pipeting. The absorbance at 570 nm (test wave length) and at 630 nm (reference wave length) was measured on an enzymelinked immunosorbent assay ELISA microplate reader MR-700 (Dynatech Corp., Frickenhausen, Germany). All assays were carried out in quadruplicate. One unit of growth-promoting activity was defined as the activity required for maximum stimulation of the growth of HL-60 cells (MT'I" absorbance was 0.5-0.6). Purification of the Growth-promoting Activity Ultrafiltration. All manipulations were done at 4~ unless otherwise specified. Thirty liters of the conditioned medium were thawed and glacial acetic acid was added at a concentration of 1%. Immediately, the acidified medium was filtered through a GA 100 glass filter. Then the medium was concentrated 100-fold by using a hollow-fiber AHP 1010 (molecular weight cutoff, 50,000) (Asahi Chemical Industry, Tokyo, Japan) and dialyzed against 10 mM acetate buffer (pH 4.0) overnight. The concentrate was centrifuged at 10,000 • g for 30 min. Protein concentration was determined with a Bio-Rad protein assay kit by the method of Bradford (6) with bovine serum albumin used as a standard. Ion-Exchange Chromatography. The dialyzed sample was applied to an SP Toyopearl cation-exchange column (1.6 • 5 cm; Tosoh Corporation, Tokyo, Japan) equilibrated with 10 mM acetate buffer, pH 4.0. The column was washed with 30 ml of the same buffer and the bound protein was eluted with a linear gradient of 0-0.8 M NaC1 in the acetate buffer. Eluted protein was detected by UV absorption at 280 nm. Reverse-Phase High-Performance Liquid Chromatography. Active fractions from the SP Toyopearl column were loaded on an Asahipak C8P-50 reverse-phase column (0.46 • 15 cm; Asahi Chemical Industry) equilibrated 3 The abbreviations used are: MTr, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS (-), Dulbecco's phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.