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Cell cycle parameters of adult rat hepatocytes in a defined medium. A note on the timing of nucleolar DNA replication

1987; Wiley; Volume: 132; Issue: 1 Linguagem: Inglês

10.1002/jcp.1041320103

1097-4652

Olav Karsten Vintermyr, Stein Ove Døskeland,

Organ Transplantation Techniques and Outcomes

Abstract Hepatocytes, isolated from adult (250–350 g) rats, attached and survived well in primary culture on highly diluted (< 1 μg/cm 2 ) 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 [ 3 H]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 (k G1/S ), 0.028/h; duration of S phase, 8.4 h; duration of G 2 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 [ 3 H]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 G 2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.