Cell cycle parameters of adult rat hepatocytes in a defined medium. A note on the timing of nucleolar DNA replication.
Hepatocytes, isolated from adult (250-350 g) rats, attached and survived well in primary culture on highly diluted (less than 1 microgram/cm2) collagen gel in a synthetic medium without serum or hormones. About 20% of the cells "spontaneously" entered S phase during the first 4 days of culturing, and mitoses were easily demonstrated at the near physiological concentration (1.25 mM) of Ca++ prevailing in the medium. Cultures given 9 nM epidermal growth factor (EGF) and 20 nM insulin 20 h after inoculation showed vigorous DNA synthesis and mitotic activity. Autoradiography of such cells exposed to [3H]thymidine allowed the determination of the following cell cycle parameters: Lag period from EGF/insulin stimulation till onset of increased DNA synthesis, 17 h; rate of entry into S phase (kG1/S), 0.028/h; duration of S phase, 8.4 h; duration of G2 phase, 2.7 h. The peak DNA synthesis (pulse labelling index, 24%) and peak mitotic activity (mitotic index, 1.7%) occurred 35 and 43 h, respectively, after the stimulation with EGF/insulin. These values are comparable to those reported during the in vivo compensatory hyperplasia following partial hepatectomy of adult rats. A marked variation of the intranuclear [3H]thymidine pulse labelling pattern was noted: During the first 1.5 h of the S phase, the labelling was extranucleolar and during the last 1.5 h chiefly nucleolar. The cells survived well in the absence of glucocorticoid, whose effect on cell cycle parameters therefore could be studied. Dexamethasone (25-250 nM) did not appreciably affect the durations of S phase and G2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.
Vintermyr
O K
OK
Døskeland
S O
SO
D000818:Animals; D002448:Cell Adhesion; D002453:Cell Cycle; D002466:Cell Nucleolus / Q000032:analysis; D003470:Culture Media; D004261:DNA Replication; D004815:Epidermal Growth Factor / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007399:Interphase; D008099:Liver / Q000166:cytology*; D008297:Male; D008938:Mitosis; D051381:Rats; D011919:Rats, Inbred Strains
D003470:Culture Media; D007328:Insulin; D004815:Epidermal Growth Factor