Summary
CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium.
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Antakly, T.; Thompson, B.; O'Donnell, D. Demonstration of the intracellular localization and up-regulation of glucocorticoid receptor by in situ hybridization and immunocytochemistry. Cancer Res. Suppl. 49:2230s-2234s; 1989.
Barnes, D. Nutritional and hormonal requirements of mammalian cell in culture. World Rev. Nutr. Diet. 45:167–197; 1985.
Barnes, D.; Sato, G. Methods for growth of cultured cells in serum-free medium. Anal. Biochem. 102:255–270; 1980.
Barnes, D.; Sato, G. Serum-free cell culture: A unifying approach. Cell 22:649–655; 1980.
Bottenstein, J.; Sato, G. Growth of a rat neuroblastoma cell line in serum-free supplemented media. Proc. Natl. Acad. Sci. USA 76(1):514–517; 1979.
Bradford, M. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254; 1976.
Brown, R.; Griffith, R.; Neubauer, R., et al. Development of a serum-free medium which supports the long-term growth of human and nonhuman primate lymphoid cells. J. Cell Physiol. 115:191–198; 1983.
Darfler, F.; Insel, P. Clonal growth of lymphoid cells in serum-free media requires elimination of hydrogen peroxide toxicity. J. Cell Physiol. 115:31–36; 1983.
Eisen, L.; Elsasser, M.; Harmon, J. Positive regulation of the glucocorticoid receptor in human T-cells sensitive to the cytolytic effect of glucocorticoids. J. Biol. Chem. 263:12044–12048; 1988.
Gersten, M.; Cohn, M. T-cell responses studied in a basal serum-free medium. Methods Enzymol. 150:129–133; 1987.
Guilbert, L.; Iscove, N. Partial replacement of serum by selenite, transferrin, albumin and lecithin in hemopoietic cell cultures. Nature 263:594–596; 1976.
Harmon, J.; Elsasser, M.; Eisen, L., et al. Glucocorticoid receptor expression in “receptorless” mutants isolated from the human leukemic cell line CEM-C7. Mol. Endocrinol. 3(4):734–743; 1989.
Harmon, J.; Norman, N.; Fowlkes, B., et al. Dexamethasone induces irreversible G1 arrest and death of a human lymphoid cell line. J. Cell Physiol. 98:267–278; 1978.
Harmon, J.; Thompson, B. Glutamine synthetase induction by glucocorticoids in the glucocorticoid-sensitive human leukemic cell line CEM-C7. J. Cell Physiol. 110:155–160; 1982.
Harmon, J.; Thompson, B. Isolation and characterization of dexamethasone-resistant mutants from human lymphoid cell line CEM-C7. Mol. Cell. Biol. 1(6):512–521; 1981.
Harmon, J.; Thompson, B.; Baione, K. Analysis of glucocorticoid-resistant human leukemic cells by somatic cell hybridization. Cancer Res. 45:1587–1593; 1985.
Harmon, J.; Schmidt, T.; Thompson, B. Molybdate-sensitive and molybdate-resistant activation-labile glucocorticoid-receptor mutants of the human lymphoid cell line CEM-C7. J. Steroid Biochem. 21(3):227–236; 1984.
Hashisume, S.; Kuroda, K.; Murakam, H. Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum free medium. Biochem. Biophys. Acta 763:377–382; 1983.
Hayashi, I.; Sato, G. Replacement of serum by hormones permits growth of cells in a defined media. Nature 259:132–134; 1976.
Higuichi, K. Cultivation of animal cells in chemically defined media, a review. Adv. Appl. Microbiol. 16:111–113; 1976.
Iscove, N.; Melchers, F. Complete replacement of serum by albumin, transferrin, and soybean lipid in cultures of lipopolysaccharide-reactive B-lymphocytes. J. Exp. Med. 147:923–950; 1975.
Lipson, S. Application of a serum-free medium in the growth and differentiation of human peripheral-blood lymphocytes. Diagn. Microbiol. Infect. Dis. 4:203–214; 1986.
McKeehan, W.; Hamilton, G.; Ham, R. Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblast. Proc. Natl. Acad. Sci. USA 73(6):2023–2027; 1976.
McDougal, J.; Kennedy, M.; Sligh, J., et al. Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule. Science 231:382–385; 1986.
McDougal, J.; Mawle, A.; Cort, S., et al. Cellular tropism of the human retrovirus HTLV-III/LAV. Nature 135(5):3151–3162; 1985.
Munck, A.; Guyre, P.; Holbrook, N. Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr. Rev. 5:25–44; 1984.
Norman, M.; Thompson, B. Characterization of a glucocorticoid-sensitive human lymphoid cell line. Cancer Res. 37:350–355; 1977.
Orly, J.; Sato, G.; Erickson, G. Serum suppresses the expression of hormonally induced functions in cultured granulosa cells. Cell 20:817–828; 1980.
Pearson, O.; Eliel, L. Use of pituitary adrenocorticotropic hormone (ATCH) and cortisone in lymphomas and leukemias. JAMA 144(16):1349–1353; 1950.
Pfahl, M.; Kelleher, R.; Bourgeois, S. General features of steroid resistance in lymphoid cell lines. Mol. Cell. Endocrinol. 10:193–207; 1978.
Ryffel, B.; Henning, C.; Huberman, E. Differentiation of human T-lymphoid leukemia cells into cells that have a suppressor phenotype is induced by phorbol 12-myristate 13-acetate. Proc. Natl. Acad. Sci. USA 79:7336–7340; 1982.
Schneider, G. The effects of preparative procedures for scanning electron microscopy on the size of isolated lymphocytes. Am. J. Anat. 146(1):93–100; 1976.
Shive, W.; Pinkerton, F.; Humpherys, J., et al. Development of a chemically defined serum and protein free medium for growth of human peripheral lymphocytes Proc. Natl. Acad. Sci. USA 83:9–13; 1983.
Shive-Matthews, K.; Pettit, F.; Boghossian, J., et al. Chemically defined medium for the growth of lymphocytes. Methods Enzymol. 150:134–146; 1987.
Soyano, A.; Romano E. Synergistic effect of albumin and transferrin on the mitogen stimulation of human mononuclear leukocytes in serum-free media. Immunol. Lett. 9:57–62; 1985.
Spicker-Polet, H.; Polet, H. Identification of albumin as a serum factor essential for the growth of activated human lymphocytes. J. Biochem. 254(2):987–992; 1976.
Tadetazu, F.; Kubota, K.; Kajigays, S., et al. Clonal growth of human acute myeloid leukemia cells (ML-1 and KL-60) in serum-free medium. Cancer Res. 44:531–535; 1984.
Thompson, B.; Harmon, J. Glucocorticoid receptors and glucocorticoid resistance in human leukemia in vivo and in vitro. In: Chrousos, G. P.; Loriaux, D. L.; Lipsett, M. B., eds. Steroid hormone resistance. New York: Plenum Publishing, 1986:111–127.
Thompson, B.; Harmon, J.; Norman, M., et al. Glucocorticoid actions in a human acute lymphoblastic leukemic, T-cell line: a model system for understanding steroid therapy. In: Roy, A. K.; Clark, J. H., eds. Hormones and cancer. New York: Raven Press; 1980:89–98.
Thompson, B.; Srivastava, D.; Johnson, B. Interactions of the phenylpyrazolo steroid cortivazol with glucocorticoid receptors in steroid-sensitive and-resistant human leukemic cells. Cancer Res. (Suppl.) 49:2253s-2258s; 1989.
Thompson, B.; Yuh, Y-S.; Ashraf, J., et al. Mechanisms of glucocorticoid function in human leukemic cells: analysis of receptor gene mutants of the activation-labile type using the covalent affinity ligand dexamethasone mesylate. J. Steroid Biochem. 30(1–6):63–70; 1988.
Thompson, B.; Yuh, Y-S.; Ashraf, J., et al. Molecular genetic analysis of glucocorticoid actions in human leukemic cells. In: Lippman, M. E., ed. Growth regulation of cancer, vol. 74. New York: Alan R. Liss; 1988:221–237.
Thompson, B.; Zawydiwski, R.; Brower, S., et al. Properties and function of human glucocorticoid receptors in steroid-sensitive and-resistant leukemic cells. In: Eriksson, H.; Gustafsson, J.-A., eds. Steroid hormone receptors: structure and function. New York: Elsevier Science Publishers; 1983:171–194.
Tormey, D.; Imrie, R.; Mueller, G. Identification of transferrin as a lymphocyte growth promotor in human serum. Cell Exp. Res. 74:163–169; 1972.
Uittenbogaart, C.; Cantor, Y.; Fahey, J. Growth of human malignant lymphoid cell lines in serum-free medium. In Vitro 19:67–72; 1983.
Vanderbilt, J.; Miesfeld, R.; Maler, B., et al. Intracellular receptor concentration limits glucocorticoid-dependent enhancer activity. Mol. Endocrinol. 1(1):68–74; 1987.
Wolff, J.; Brubaker, C.; Murphy, M., et al. Prednisone therapy of acute childhood leukemias—prognosis and duration of response in 330 treated patients. J. Pediatr. 70:626–631; 1967.
Yen, A.; Duigou, R. Serum-free media for a human lymphocyte cell line and for PMA-stimulated peripheral blood lymphocytes: requirements for insulin, transferrin and albumin. Immunol. Lett. 6:169–179; 1983.
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This work was conducted in conjunction with the Walls Medical Research Foundation.
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Chilton, D.G., Johnson, B.H., Danel-Moore, L. et al. Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media. In Vitro Cell Dev Biol 26, 561–570 (1990). https://doi.org/10.1007/BF02624204
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DOI: https://doi.org/10.1007/BF02624204