STEM-CELL FACTOR AND STROMAL CELL COCULTURE PREVENT APOPTOSIS IN A SUBCULTURE OF THE MEGAKARYOBLASTIC CELL-LINE, UT-7

The megakaryoblastic cell line, UT-7, is dependent for its growth upon interleukin-3 (IL-3), erythropoietin, or granulocyte-macrophage colon y stimulating factor (GM-CSF). A subculture of this line can be mainta ined in recombinant human c-kit ligand [stem cell factor (SCF)] at 100 ng/ml without requirement for other growth factors. Removal of this s ubculture from SCF results in rapid loss of viability and decreased pr oliferation. Cells grown in SCF also can be maintained in GM-CSF but n ot vice versa. In this work, we have characterized the SCF dependence of this UT-7 subculture. Stem cell factor removal results in apoptosis and a decline in viability which can be restored partially by re-addi tion of SCF, GM-CSF, or co-culture with adherent marrow stromal cells. Apoptosis in the factor-starved UT-7 population has been documented b y light microscopy, electron microscopy and DNA analysis, showing the typical 180 base pair laddering characteristic of apoptosis. To quanti tate the degree of apoptosis in the cell populations, and to assess wh ether apoptosis decreased with re-exposure of starved cells to growth factors or stroma, we utilized flow cytometry. This confirmed that exp osure of previously factor-starved cells to stroma decreased the perce ntage of cells undergoing apoptosis. Co-culture with an SCF-deficient murine stromal cell line was also able to prevent apoptosis, suggestin g contribution of other stromal cell factors. Experiments performed us ing trans-well inserts which do not allow cell passage, showed greates t viability of cells in contact with stroma, but viability was also im proved in cells cultured in the presence of, but not in contact with, stromal cells compared to those cultured above plastic, suggesting a r ole for soluble stroma-produced substances. These data demonstrate tha t SCF alone can prevent apoptosis in cells dependent upon its presence for proliferation. Also, marrow stromal cells can serve as a partial substitute for growth factor in the prevention of apoptosis in these c ells, probably due to constitutive presentation of SCF and other hemat opoietic growth factors in both soluble and surface-bound forms. Copyr ight (C) 1996 Elsevier Science Ltd.