Growth and macromolecular synthesis in the 3T6 mouse fibroblast. I. General description and the role of ascorbic acid.

The growth of the j T6 mouse fibroblast and synthesis of macromolecules has been studied over the last 6 days of the cultures' 14-day life span. The effect of ascorbic acid was also tested. During this period, the cells synthesized collagen, as judged by the appearance of nondialysable hydroxyproline, which was identified by chemical assay and by radioactive incorporation studies. A high proportion of the collagen in the cell layer was insoluble ino-iN acetic acid. Of the hydroxyproline synthesized in the presence of ascorbic acid, about 75 % was eventually liberated into the growth medium, and about 25-30% of the liberated material behaved as free hydroxyproline. In the absence of ascorbic acid, the cell layer hydroxyproline was reduced to one-third, but the growth medium hydroxyproline was unaffected. The cells also synthesized glycosaminoglycans, as judged by the appearance of cetyl pyridinium-precipitable uronic acid, and the incorporation of labelled glucosamine into macromolecules. A large proportion of this material has the properties of hyaluronic acid. Ascorbic acid had no detectable effect on overall glycosaminoglycan synthesis, in contrast to healing tendonectomy wounds in guinea-pigs. Cell proliferation and general protein synthesis were virtually unaffected by ascorbic acid. Whereas general protein synthesis, like cell proliferation, declined in the ageing culture, glycosaminoglycan synthesis and collagen synthesis continued at a steady or increasing rate.