Abstract
Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma.
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Associan RK, Komoriya A, Meyers CA, Miller DM and Sporn MB (1983) Transforming growth factor-β in human platelets. J. Biol. Chem. 258: 7155–7160.
Denel TF, Huang JS, Proffitt RT, Baeuzger JU, Chang D and Kennedy BB (1981) Human platelet-derived growthfactor purification and resolution into 2 active protein fractions. J. Biol. Chem. 256: 8896–8899.
Hashizume S, Kuroda K and Murakami H (1983) Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium. Biochim. Biophys. Acta 258: 7155–7160.
Hashizume S, Murakami H, Hirose S, Shirai T, Shinohara K and Omura H (1986) Human-human hybridomas from lung cancer patients secrete anti-DNA antibodies. Agric. Biol. Chem. 50: 1351–1353.
Hashizume S, Murakami H, Kamei M, Hirose S, Shirai T, Yamada K and Omura H (1987) Specificity of anti-polynucleotide monoclonal antibodies from human-human hybridomas. In Vitro Cell. Develop. Biol. 23: 53–56.
Heldin CH, Westermark B and Wasteson A (1981) Plateletderived growth factor. Biochem. J. 193: 907–913.
Köhler G and Milstein C (1975) Continuous cultures of fused cells secreting antibodies of predefined specifity. Nature 256: 495–497.
Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227: 680–685.
Miyata M, Hashizume S, Murakami H, Yamada K and Omura H (1988) Characterization and application of lymphocytic clonal growth factor in human plasma. Cytotechnology 1: 233–241.
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxity assays. J. Immunol. Methods 65: 55–63.
Murakami H, Masui H, Sato GH, Sueoka N, Chow TP and Kano-Sueoka T (1982) Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component. Proc. Natl. Acad. Sci., USA 79: 1158–1162.
Murakami H, Hashizume S, Ohashi H, Shinohara K, Yasumoto K, Nomoto K and Omura H (1985) Human-human hybridomas secreting antibodies specific to human lung carcinoma. In Vitro Cell. Develop. Biol. 21: 593–596.
Murakami H (1987) Production of monoclonal antibodies with human-human hybridomas in serum-free culture and its prospects for medical use. In: Umeda et al., (ed) Biotechnology of Mammalian Cells (pp. 119–136) Japan Sci. Soc. Press, Tokyo, Springer Verlag, Berlin.
Murakami H, Okazaki Y, Yamada K and Omura H (1988) Egg yolk lipoprotein, a new supplement for the growth of mammalian cells in serum-free medium. Cytotechnology 1: 159–169.
Nagata S, Tsuchiya M, Asano S, Kaziro Y, Yamazaki T, Yamada O, Hirata Y, Kubota N, Oheda M, Nomura H and Ono M (1986) Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor. Nature 319: 415–418.
Ohara J, Lahet S, Inman J and Paul W (1985) Partial-purification of murine B-cell stimulatory factor (BSF-1). J. Immunol. 135: 2518–2523.
Ohashi H, Hashizume S, Murakami H, Aihara K, Shinohara K and Omura H (1986) HO-323, a human B-lymphoblastoid cell line useful for making human-human hybridomas. Cell Biol. Interntl. Rep. 10: 77–83.
Shinmoto H, Murakami H, Dosako S, Shinohara K and Omura H (1986) Human-human hybridomas secreting IgM-like immunoglobulin with α and μ heavy chains. Agric. Biol. Chem. 50: 2217–2223.
Shinmoto H, Murakami H, Yamada K, Dosako S and Omura H (1988) Immunoglobulin production stimulating and inhibiting factors derived from a human lung adenocarcinoma PC-8 cells. Cytotechnology 1: 295–300.
Takazawa Y, Tokashiki M, Murakami H, Yamada K, and Omura H (1988) High-density culture of mouse-human hybridoma in serum-free defined medium. Biotechnol. Bioeng. 31: 168–172.
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Miyata, M., Murakami, H., Hashizume, S. et al. Purification and characterization of lymphocytic clonal growth factor (LCGF) derived from human-human hybridoma SH-76 cells. Cytotechnology 1, 347–353 (1988). https://doi.org/10.1007/BF00365080
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DOI: https://doi.org/10.1007/BF00365080