DNA polymerase and thymidine kinase activities in MC-29 virus-induced transplantable hepatoma and the effect of cytostatic treatment on these activities

Abstract

The activity of DNA polymerases and thymidine kinase was compared in the MC-29 leukosis virus-induced transplantable hepatoma and in the livers of rats treated with cyclophosphamide (CP), cytosine-arabinoside (ara-C) and 5-fluoro-uracil (5-FU). The specific activity of DNA polymerase was twenty times greater in the MC-29 leukosis virus-induced hepatoma, while thymidine kinase was only 3–5 times greater than in the liver. All three enzymes showed Michaelis-Menten kinetics in their substrate and template saturation curves. The template utilization of DNA polymerases from hepatoma and from liver was compared. Both had higher activities on a poly(dA) · poly(dT) template at pH 8.0, than on DNA at pH 7.5. After chromatography on a phosphocellulose column two polymerases were separated. The first peak eluted by 0.15 M KCl preferred DNA as template (polymerase α). The second eluted by 0.5 M KCl worked better on poly(dA) · poly(dT) (polymerase β). Thymidine kinase was eluted by 0.25 M KCl. Inhibition by N-ethylmaleimide (NEM) showed the polymerase α to be sensitive and the polymerase β to be resistant to the sulfhydryl blocking agent; similar to the respective enzymes of other eukaryotic cells. The specific acitivity of DNA polymerase decreased after CP treatment at 6 h and 72 h and after ara-C treatment at 72 h. The specific activities of thymidine kinase were not changed significantly in response to the drug administrations.