| Abstract: | Chinese hamster cells (line CHO) maintained in McCoy's 5A medium (modified) supplemented with insulin (10 micrograms/ml), transferrin (5 micrograms/ml), and ferrous sulfate (1.1 microgram/ml) proliferate at rates similar to cultures growing in the McCoy's medium supplemented with 10% fetal bovine serum. Colony-forming ability is similar in cultures supplemented with either serum or the combination of growth factors. By 6 hours after replacement of serum with growth factors, ornithine decarboxylase (ODCase) activity increases, reaching a maximum value by 24 hours after serum replacement. This maximum is cell density dependent and can exceed a 30-fold increase over enzyme activity in cultures supplemented with serum. The increased enzyme activity is due to a decrease in the turnover rate of the enzyme, based on protein synthesis inhibition studies, and an accumulation of active enzyme molecules rather than an activation of existing molecules, since the catalytic activity of ODCase, determined using the radiolabeled form of alpha-difluoromethylornithine (an enzyme-activated, irreversible inhibitor of ODCase) in concert with supplements. Intracellular putrescine and spermidine levels are substantially decreased when cultures are maintained in medium supplemented with insulin, transferrin, and ferrous sulfate, rather than serum, which is the sole source of exogenous ornithine. Titration of cultures growing in the defined medium with ornithine leads to a decrease in ODCase activity and an increase in intracellular putrescine and spermidine levels. Putrescine- and spermidine-dependent S-adenosyl-L-methionine decarboxylase activities are similar in cultures maintained in either medium. These data demonstrate that some, but not all, aspects of polyamine biosynthesis are affected by the availability of ornithine, the first substrate in the pathway. |
