Primary explants of P388, EL-4, and L1210 murine leukemia cells and of normal mouse bone marrow are shown to require sulfhydryl compounds for proliferation in vitro. Nine established cell lines show no stimulation by these compounds. Leukemia cells can lose the sulfhydryl dependence after various periods of adaptation to in vitro culture. Various sulfhydryl compounds have widely differing potencies in promoting in vitro proliferation of dependent cells. The effect appears to be specific for sulfhydryl groups in the reduced form. Vitamin B12 compounds inhibit the growth of sulfhydryl-requiring cells, apparently by catalyzing the oxidation of the sulfhydryl groups.Sulfhydryl compounds have received relatively little attention as essential nutrients for cells in vitro. In 1971, Park et al. (1) briefly described stimulation by cysteine and glutathione of plasmacytoma growth in vitro. Broome and Jeng, in 1972, reported a requirement for sulfhydryl compounds in certain murine leukemia cells in vitro (2, 3). Other reports describe enhancement by sulfhydryl compounds of blast transformation (4, 5), of antibody formation (6-9), and of viability (2,8) in lymphocyte cultures. This report describes a study of the sulfhydryl dependence of some hematopoietic cells and the inhibitory effect of vitamin B12 compounds on these cells.
MATERIALS AND METHODSCell Culture. Eagle's minimum essential medium (MEM, ref. 10) was supplemented with 5-20% fetal calf serum (as indicated), 30 mg of glutamine/100 ml, 5 mg of asparagine/100 ml, 10 mg of serine/100 ml, and 11 mg of sodium pyruvate/100 ml. No antibiotics were added. In experiments involving a series of variable additives, a single homogeneous culture was used to pour all plates in the series. Variable additives were sterilized by filtration and placed in the plastic culture dishes before the culture was added.P388 murine leukemia cells (11) and L1210 murine leukemia cells (12) were obtained from peritoneal aspirates of DBA/2 mice which were inoculated 4-6 days previously with 106 cells. EI-4 murine leukemia cells (13) were obtained in a similar manner from C57 mice. Leukemia cells were cultured in 1.5-ml cultures in liquid medium in 35 X 10-mm Falcon plastic plates with an initial cell density of 1 X 105 cells per ml. After incubating at 370 in 5% CO2 for the indicated number of days (usually 3 days), the cells were suspended by swirling and diluted either 1 to 2 in trypan blue for counting in a hemocytometer or 1 to 800 in Isoton for counting in a Coulter counter.The For bone marrow culture, the medium was made at two times the normal concentration and mixed with an equal volume of 0.6% agar at 370 before adding fresh bone marrow cells obtained from the femurs of white Swiss Webster mice. Colony-stimulating factor was provided as an extract of gravid C3H or DBA/2 mouse uterus prepared by the method of Bradley et al. (21). Cultures of 1.5 ml were plated as a single layer in 35 X 10-mm Falcon plastic plates. Clones of more than 20 cells were counted as colonies on the seve...