Hyaluronic acid synthetase in cultured mammalian cells producing hyaluronic acid Oscillatory change during the growth phase and suppression by 5-bromodeoxyuridine

Abstract

A hybrid line B-6 synthesizes and secretes a large amount of hyaluronic acid and this differentiated function is suppressed by growth in the presence of the thymidine analog, 5-bromodeoxyuridine. In order to elucidate the mechanism of this analog inhibition, the levels hyaluronic acid synthetase and UDPglucose dehydrogenase in the cells were investigated. The results obtained were as follows. 1.1.The addition of 5-bromodeoxyuridine at concentrations as low as 5 μg/ml to medium while having the effect on cell growth, caused a great drop in the activity of hyaluronic acid synthetase. By contrast, the specific activity of UDPglucose dehydrogenase was slightly increased.2.2. This suppression on the synthetase was detected 10 h after addition of the analog; when the culture treated for one generation (16 h) was transferred to the analog-free medium, the activity was recovered to control value during the next three to four generations. The analog effect was prevented by simultaneous addition of hydroxyurea, an inhibitor of DNA synthesis, with the analog. These results prove that incorporation of the analog into DNA is necessary for this inhibitory effect.3.3. In the presence of either actinomycin D or cycloheximide, cellular hyaluronic acid synthetase activity decayed with a half-life of 3.5 and 2 h, respectively, whereas UDPglucose dehydrogenase decayed with a half-life of 20 h.4.4. The activity of the synthetase, but not that of UDPglucose dehydrogenase, was correlated with high growth rate, falling sharply with decline of cell growth. Recovery from low activity in the cells transferred to fresh medium required new synthesis of both RNA and protein. 1.The addition of 5-bromodeoxyuridine at concentrations as low as 5 μg/ml to medium while having the effect on cell growth, caused a great drop in the activity of hyaluronic acid synthetase. By contrast, the specific activity of UDPglucose dehydrogenase was slightly increased. 2. This suppression on the synthetase was detected 10 h after addition of the analog; when the culture treated for one generation (16 h) was transferred to the analog-free medium, the activity was recovered to control value during the next three to four generations. The analog effect was prevented by simultaneous addition of hydroxyurea, an inhibitor of DNA synthesis, with the analog. These results prove that incorporation of the analog into DNA is necessary for this inhibitory effect. 3. In the presence of either actinomycin D or cycloheximide, cellular hyaluronic acid synthetase activity decayed with a half-life of 3.5 and 2 h, respectively, whereas UDPglucose dehydrogenase decayed with a half-life of 20 h. 4. The activity of the synthetase, but not that of UDPglucose dehydrogenase, was correlated with high growth rate, falling sharply with decline of cell growth. Recovery from low activity in the cells transferred to fresh medium required new synthesis of both RNA and protein.