Tyrosine aminotransferase sensitivity to bromodeoxyuridine during restricted intervals of S phase in hepatoma cells.

Abstract

Synchronized hepatoma tissue culture (HTC) cells, accumulated at the G,/S boundary with aminopterin, were released into S phase with either thymidine or 5-bromodeoxyuridine (BUdR) . Tyrosine aminotransferase (TAT) activity was found to be unaffected by BUdR over the initial 3 h of S phase, but then to rapidly decline to a new basal level of 40% of control by 9 h . There was no corresponding response in the activities of alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and alkaline phosphatase, or in the rate of protein and RNA synthesis. If BUdR incorporation was restricted to limited periods of S phase, TAT was found to be maximally suppressed by incorporation into the initial 40% of the DNA . Incorporation of the analogue into the latter 60% of DNA synthesized during S phase had no effect on TAT. This is the first report that the effect of BUdR on TAT in HTC cells is associated with incorporation of the analog into DNA synthesized during a specific interval of S phase.The thymidine (dThd) analogue 5-bromodeoxyuridine (BUdR) has been found to be a unique modulator of eukaryotic gene expression . Its incorporation into DNA is associated with blocked differentiation, the induction of viral particles, and either the suppression or stimulation of specific functions in terminally differentiated cell types (for review, see Goz [7]) .In attempts to understand the mechanism(s) of action of BUdR, one of the most extensively studied systems is tyrosine aminotransferase (TAT) in hepatoma tissue culture (HTC) cells . Stellwagen and Tomkins (19,20) reported that growth of HTC cells in BUdR led to a rapid decrease in TAT activity that could not be attributed to a soluble inhibitor, a defective enzyme, or a change in enzyme degradation . Within the time period studied, there was no effect on other enzymes, cell proliferation, or protein and RNA synthesis. The analogue affected TAT only if present during S phase, and O'Brien and Stellwagen (16) found the TAT decrease was proportional to the percent of dThd residues replaced by BUdR in each new strand of DNA . Stellwagen and Tomkins (19) proposed that BUdR blocked transcription of the TAT structural gene .The S phase of the eukaryotic cell is envisioned as being a synchronous period within itself during which genes replicate in repetitive sequence from one S phase to another (for review, see Hand [8]) . If the TAT gene exists as a unique, single copy of DNA, and BUdR exerts its effect only when in the structural gene, then the effect should be limited to the incorporation of BUdR during a specific interval of S phase . That interval should be the time during which the effected gene replicates .