Basis for natural variation in sensitivity to 5-fluorouracil in mouse and human cells in culture.

Abstract

Abstract Seven established human cell lines in vitro were, on the average, 8 times less sensitive to growth inhibition by 5-fluorouracil (FUra) than were eight mouse cell lines. Four of the human cell lines (U-cells, HeLa, KB, and Hep-2) and three mouse cell lines (S-180, MMT, and LM) were examined to clarify the basis of the different sensitivities. The K m 9s and V max 9s for the total cellular uptake of FUra were higher in the more sensitive (K m 300 to 500 µm; V max 110 to 140 pmol/min/mg cellular protein) than in the less sensitive cells (K m 30 to 120 µm; V max 10 to 70 pmol/min/mg cellular protein). At a fixed concentration of [2- 14 C]FUra (10 µm), the velocity of incorporation of the analog into acid-soluble and -insoluble material was up to 7 times more rapid in the more sensitive cells. The major acid-soluble metabolite was 5-fluorouridine triphosphate, which after 3 hr incubation with 10 µm FUra comprised 60 to 90% of soluble radioactivity and reached 43 to 130 µm in the cell water of the more sensitive and 11 to 24 µm in the less sensitive cells. No acidsoluble 5-fluorodeoxyuridine-5′-monophosphate was detected. There was no difference in the velocity of accumulation of unaltered FUra. This suggested that the conversion of FUra to nucleotides was slower in the less sensitive cells. Of three enzymes (uridine phosphorylase, thymidine phosphorylase, and FUra phosphoribosyltransferase), at least two had higher activities in the extracts of the more sensitive cells. There was no significant difference in fluorouridine kinase activity, while the activity of thymidine kinase was consistently higher in the less sensitive cells. These findings show that sensitivity to FUra of cells in vitro , whether of mouse or human origin, is associated with: ( a ) greater activity of enzymes responsible for converting FUra to 5-fluorodeoxyuridine, 5-fluorouridine, and 5-fluorouridine-5′-monophosphate; ( b ) lower activity of thymidine kinase; and ( c ) more rapid intracellular accumulation of acid-soluble FUra nucleotides and their incorporation into RNA.