Relationship between DNA topoisomerase I activity and DNA synthesis in cultured hepatocytes: effects of orotic acid.
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.
Giannoni
P
P
Institute of General Physiology, University of Genoa, Italy.
Fronza
G
G
Scarabelli
L
L
Fugassa
E
E
Orunesu
M
M
Cesarone
C F
CF
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
D009963:Orotic Acid; D004815:Epidermal Growth Factor; D004264:DNA Topoisomerases, Type I