Stem cell factor and stromal cell co-culture 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 colony stimulating factor (GM-CSF). A subculture of this line can be maintained in recombinant human c-kit ligand [stem cell factor (SCF)] at 100 ng/ml without requirement for other growth factors. Removal of this subculture from SCF results in rapid loss of viability and decreased proliferation. Cells grown in SCF also can be maintained in GM-CSF but not 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-addition of SCF, GM-CSF, or co-culture with adherent marrow stromal cells. Apoptosis in the factor-starved UT-7 population has been documented by light microscopy, electron microscopy and DNA analysis, showing the typical 180 base pair laddering characteristic of apoptosis. To quantitate the degree of apoptosis in the cell populations, and to assess whether apoptosis decreased with re-exposure of starved cells to growth factors or stroma, we utilized flow cytometry. This confirmed that exposure of previously factor-starved cells to stroma decreased the percentage of cells undergoing apoptosis. Co-culture with an SCF-deficient murine stromal cell line was also able to prevent apoptosis, suggesting contribution of other stromal cell factors. Experiments performed using trans-well inserts which do not allow cell passage, showed greatest viability of cells in contact with stroma, but viability was also improved in cells cultured in the presence of, but not in contact with, stromal cells compared to those cultured above plastic, suggesting a role for soluble stroma-produced substances. These data demonstrate that 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 cells, probably due to constitutive presentation of SCF and other hematopoietic growth factors in both soluble and surface-bound forms.
Liesveld
J L
JL
Department of Medicine, University of Rochester School of Medicine, NY 14642, USA. Jane@bmt.roch.edu
Harbol
A W
AW
Abboud
C N
CN
D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001854:Bone Marrow Cells; D003994:Bucladesine / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival; D018920:Coculture Techniques; D004273:DNA, Neoplasm / Q000032:analysis; D004921:Erythropoietin / Q000494:pharmacology; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006133:Growth Substances / Q000276:immunology / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D007700:Kinetics; D007947:Leukemia, Megakaryoblastic, Acute / Q000473:pathology*; D008854:Microscopy, Electron; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology*; D017154:Stromal Cells / Q000502:physiology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000906:Antibodies; D004273:DNA, Neoplasm; D006133:Growth Substances; D007377:Interleukin-3; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D004921:Erythropoietin; D003994:Bucladesine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor