We have studied the kinetics of suppression of tyrosinase activity and tumorigenicity in unsynchronized B16 mouse melanoma cells (clone B559) exposed to 5-bromodeoxyuridine (BrdU, 3 mug/ml) for one or two cell divisions, then cultured in BrdU-free medium (RM) for five or six days. Bromouracil replaced about 23% of thymine residues after 24 hours (1 cell division) and almost 40% after 48 hours (2 cell divisions) in the presence of BrdU. Upon subsequent growth in RM the extent of replacement declined in a manner consistent with dilution by new DNA synthesis, reaching 5-10% substitution by day 7 of these experiments. Tyrosinase activity was significantly reduced after treatment with BrdU for 24 or 48 hours but continued to decline after the cultures were changed to RM, approaching undetectable levels on day 7. The time course of reduction was similar to that previously determined in cells grown continuously for seven days in the presence of BrdU. Therefore, suppression of tyrosinase activity can result from incorporation of BrdU during a single cell cycle, but requires about seven days for full manifestation of the effect. Tumorigenicity decreased to 55% after 24 hours and to 15% after 48 hours with BrdU but rapidly reversed to approach that of untreated melanoma cells when subsequently grown in RM for 5-6 days. The effects of BrdU on total RNA or protein synthesis, or on plating efficiency appeared insufficient to account for the degree of suppression observed. Our results indicate that substitution by bromouracil into either strand of DNA loci controlling tyrosinase activity or tumorigenic potential may be sufficient for suppression. In addition, they demonstrate that such brief treatment with BrdU may be used to probe the regulation of differentiated function and tumorigenicity in these melanoma cells.
