DNA ligase activities during hepatocarcinogenesis induced by N-2-acetylaminofluorene

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Abstract
A progressive accumulation of DNA breaks has been reported to occur in nuclear DNA obtained from putative premalignant hepatic lesions induced by carcinogens. To determine if this alteration resulted from a defect in the level of, or functional activity of DNA ligases, we compared these enzymes in normal rat liver, 24-h regenerating liver, and hepatic nodules at intervals after cessation of N-2-acetylaminofluorene (AAF) treatment. Nuclear extracts of hepatocytes were separated into soluble and chromatin fractions, and multiple forms of DNA ligase activity were obtained by AcA34 gel filtration chromatography. The activities of the two largest species, DNA ligase la (480 kd) and DNA ligase Ib (240 kd), were present exclusively in soluble, nuclear fractions and were increased 4-fold and 2-fold, respectively, in 24-h regenerating livers. In AAF-induced nodules, these species were increased 3-fold and 1.5-fold, respectively, above those of normal rat liver, somewhat higher than predicted from the rate of cell division. In all of the test tissues, these ligase species demonstrated identical sensitivity to inhibition with 0.1 M NaCl or heating at 50°C. DNA ligase II (80 kd) was found in both soluble nuclear fractions and chromatin at approximately identical levels in all tissues tested. Ligase II from all tissues also demonstrated identical responses to salt and heat. These data support the concept that DNA ligases la and Ib are related to DNA replication and suggest that ligase II may be a repair enzyme. The failure to detect significant alterations from expected values in the hepatic nodules and the lack of alteration in sensitivity to salt and heat indicate that the accumulation of DNA damage (presumably breaks) previously observed in carcinogen-induced altered hepatocytes is not due to an alteration in the level or the biochemical properties of DNA ligase.