The nature of 5-bromodeoxyuridine inhibition of muscle enzyme development in vitro

Abstract Although it is generally believed that 5-bromodeoxyuridine (BrdUrd) can suppress the phenotypic expression of a wide variety of cell types by virtue of its incorporation into DNA, clear quantitative evidence has been lacking. We have employed the enzymes (creatine phosphokinase and glycogen phosphorylase) which are important in energy provision for contraction and linked with the morphological differentiation of muscle, as markers for examining the mechanism of BrdUrd inhibition. Several findings lead us to conclude that BrdUrd most likely prevents the synthesis of these enzymes as a result of its presence in DNA: (1) BrdUrd is most effective as an inhibitor while mononucleated muscle cells are synthesizing DNA; (2) the reversal of inhibition requires times of the order of a division(s) and is 5-fluorodeoxyuridine (FdUrd) sensitive; (3) loss of enzyme activity can be correlated with increasing [ 3 H]BrdUrd incorporation into DNA; and (4) altering the extent of [ 3 U]BrdUrd incorporation into DNA by addition of thymidine also decreases the inhibitory effectiveness of BrdUrd. As noted by others, BrdUrd appears to preferentially inhibit those enzymes which are usually associated with muscle. In addition, we find from our studies with BrdUrd that the development of these enzymes and myotube formation may be separate events. There are no apparent altered forms of other enzymes produced in BrdUrd treated cells. In muscle, BrdUrd does not appear to act by exclusively inhibiting inducible enzymes. The manner by which BrdUrd appears to preferentially inhibit “tissue specific” products can still only be treated speculatively. However, the muscle isoenzymes of creatine phosphokinase and glycogen phosphorylase serve as experimentally amenable markers for probing the molecular details involved in BrdUrd action.