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Stimulation of DNA Synthesis and Thymidine Kinase Activity in Human Embryonic Kidney Cells Infected by Adenovirus 2 or 12¹

Putnam Memorial Hospital Institute for Medical Research, Bennington, Vermont 05201

Secondary cultures of human embryonic kidney cells were infected with Adenovirus Type 2 or Type 12. The formation of infective virus was first detected approximately 18 hours after Adenovirus 2 infection, and 20–22 hours after Adenovirus 12 infection. The total intracellular infective titer of Adenovirus 2 increased until approximately 40 hours after infection, at which time 2400–3100 plaque-forming units per cell were produced. The intracellular infective titer of Adenovirus 12, on the other hand, increased until about 65 hours after infection, when 240–390 plaque-forming units were obtained per cell.

The phosphorylation of thymidine-³H (thymidine kinase activity) was enhanced after infection by either Adenovirus 2 or 12. Increased enzyme activity was first detected about 19 hours after infection with either virus, and at 30 to 50 hours postinfection, the activity was four- to six-fold higher than that observed in the uninfected cells. The presence of puromycin in cultures during the 19- to 30-hour postinfection period inhibited the increase in enzyme activity. Removal of the puromycin at 30 hours did not restore thymidine kinase activity. Actinomycin D prevented thymidine kinase induction when added to cultures shortly after infection with Adenovirus 2 or 12 and partially arrested the additional increase in enzyme activity when added after enzyme induction had begun. Extracts prepared from cells infected by either adenovirus did not activate the thymidine kinase of extracts from uninfected cells, nor did the uninfected cell extracts inhibit the enzyme activity of the infected cell extracts.

Several properties of the thymidine kinase were studied in crude extracts prepared from uninfected cells and from cells infected by Adenovirus 2 or 12. No appreciable differences between the enzyme extracts were found with respect to sensitivity to feedback inhibition by deoxythymidine-5'-triphosphate, thermostability, or K_m values.

At approximately the time that the increase in thymidine kinase activity occurred in the infected cells, there was a significant stimulation of the rate of incorporation of the radioactive precursor, thymidine-³H, into the DNA of cells infected by either Adenvoirus 2 or 12.

¹ This work was supported by Damon Runyon Grant 882A(T).

² The author is a recipient of a research career development award, 1-K3-CA-5278-01, from the National Cancer Institute, USPHS.

Received 1/11/67. Accepted 4/18/67.