HeLa cells resistant to bromodeoxyuridine and deficient in thymidine kinase activity

Mutant sublines of HeLa S3 cells resistant to growth inhibition by bromodeoxyuridine (BUdR) have been isolated. The resistant cell lines (HeLa BU-10, HeLa BU-15, HeLa BU-25, HeLa BU-50, and HeLa BU-100) proliferated in the presence of 10, 15, 25, 50, and 100 μg/ml BUdR, respectively. Extracts from HeLa BU-25, HeLa BU-50, and HeLa BU-100 cells exhibited 2–5 per cent of the thymidine-3H, deoxyuridine-3H, and bromodeoxyuridine-3H phosphorylating activities of parental HeLa S3 cells. HeLa BU-10 and HeLa BU-15 cell extracts were also deficient in thymidine kinase activity, yielding approximately 43 per cent and 8 per cent, respectively, the thymidine kinase activity of parental HeLa S3 cells. The deficiency in thymidine kinase activity of HeLa BU-100 cells was not due to negative feedback inhibition by high levels of BUdR or to interference with the thymidine kinase assay by inhibitors or competing enzymes in the HeLa BU-100 cell extracts. Following 5 weekly passages in media lacking BUdR, the HeLa BU-100 cells did not exhibit increased thymidine kinase activity. Moreover, mixtures of extracts from HeLa S3 and HeLa BU-100 cells displayed a thymidine kinase activity equivalent to the sum of the activities of extracts prepared, respectively, from the HeLa S3 and HeLa BU-100 cells. Radioautographic studies have shown that after HeLa S3 cells were incubated for 6 hours with thymidine-3H, 35–45 per cent of the nuclei were heavily labeled with radioactivity. However, fewer HeLa BU-100 cells displayed labeled nuclei and the nuclei were only lightly labeled. HeLa BU-100 cell extracts contained normal amounts of thymidylate synthetase, thymidylate kinase, and uridine kinase activities. Following infection by vaccinia virus, high levels of thymidine kinase activity were induced in HeLa BU-100 cells. La deficience de l'activite thymidine-kinasique des cellules HeLa BU-100 n'etait pas due a une inhibition du feedback negatif par de fortes concentrations de BUdR ni a une perturbation du dosage de la thymidine kinase par des enzymes inhibiteurs ou concurrents dans les extraits de cellules HeLa BU-100. En effet, apres cinq passages hebdomadaires dans des milieux prives de BUdR, les cellules HeLa BU-100 ne montraient aucun accroissement de l'activite thymidine kinasique. En outre, les melanges d'extraits de cellules/HeLa S3 et HeLa BU-100 presentaient une activite thymidine kinasique equivalant a la somme des activites des extraits prepares a partir des cellules HeLa S3 et HeLa BU-100 respectivement. L'observation autoradiographique a montre que, lorsque les cellules HeLa S3 etaient mises en incubation pendant 6 heures avec de la thymidine-3H, 35 a 45 pour cent des noyaux etaient fortement marques par la radioactivite. Par contre, les noyaux marques etaient plus rares dans les cellules HeLa BU-100 et ceux qui etaient marques ne l'etaient que legerement. Les extraits de cellules HeLa BU-100 presentaient des activites normales de la thymidylate synthetase, de la thymidylate kinase et de l'uridine kinase. L'infection des cellules HeLa BU-100 par le virus de la vaccine a suscite une forte activite de la thymidine kinase.