title: Relationship between DNA topoisomerase I activity and DNA synthesis in cultured hepatocytes: effects of orotic acid.
abstract: Previous studies have demonstrated that DNA topoisomerase I activity can be closely related to DNA replication and active transcription in different experimental models. This relationship was further investigated by studying the time course of DNA topoisomerase I activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. DNA topoisomerase I activity was assayed quantifying ATP-independent relaxation of a negatively supercoiled plasmid, substrate for the enzyme. Incubation of cellular extracts was carried out at 30 degrees C for 15'. Products were electrophoresed and analyzed by densitometry. DNA synthesis was measured as [3H]thymidine incorporation in the hepatocytes, after a 2 hrs pulse labelling. An increase in DNA topoisomerase I activity was observed early after epidermal growth factor addition, before the onset of DNA synthesis. Concomitant administration of orotic acid, which has recently shown mitoinhibitory effect, abolished the EGF-induced activity as well as DNA synthesis. Taken together these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by DNA topoisomerase I with a precise time schedule. Orotic acid administration abolishes this response and causes an overall loss in DNA topoisomerase I activity.
authors.affiliation: Institute of General Physiology, University of Genoa, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Giannoni
authors.forename: G
authors.initials: G
authors.lastname: Fronza
authors.forename: L
authors.initials: L
authors.lastname: Scarabelli
authors.forename: E
authors.initials: E
authors.lastname: Fugassa
authors.forename: M
authors.initials: M
authors.lastname: Orunesu
authors.forename: C F
authors.initials: CF
authors.lastname: Cesarone
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004261:DNA Replication / Q000187:drug effects*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D009963:Orotic Acid / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D009963:Orotic Acid; D004815:Epidermal Growth Factor; D004264:DNA Topoisomerases, Type I