TGF-beta completely blocks the formation of small-cell colonies: effects of mito-inhibitory factors on the proliferation of primary cultured rat hepatocytes.
The proliferation of primary cultured rat hepatocytes was observed in serum-free modified DMEM supplemented with 10 mM nicotinamide and 10 ng/ml EGF. These proliferating cells were mainly mononucleate and formed small-cell colonies after 4 days of culture. In the present experiment primary cultured hepatocytes were treated with Activin A, IL-1 beta, IL-6, and TGF-beta, which have been shown to be inhibitors of the DNA synthesis of rat hepatocytes, to examine whether these four inhibitors could suppress the formation of small-cell colonies. The initial DNA synthesis of more than 50% of the cells was dose-dependently inhibited by all the factors and the strongest inhibition was demonstrated in the cells treated with TGF-beta. Although Activin A and IL-6 did not block the colony development when the agents were administered at 96 h, just before the time when the cells started to form colonies, TGF-beta and IL-1 beta could inhibit the colony formation completely and partially, respectively. Transient treatment (48-72 h) with TGF-beta was enough to suppress colony development, while Activin A and IL-6 did not block the formation of colonies. IL-1 beta partially suppressed this formation. However, continuous administration (48-144 h) of IL-beta as well as TGF-beta stimulated the detachment of the cells from dishes and the remaining hepatocytes failed to form colonies. In addition, only TGF-beta could inhibit the DNA synthesis of most small cells in the established colonies as well as that of relatively large hepatocytes. Neither Activin A, IL-1 beta nor IL-6 could inhibit the DNA synthesis of the small cells. Thus, only TGF-beta could completely inhibit both the DNA synthesis of any type of hepatocyte and the formation of small-cell colonies.
Mitaka
T
T
Department of Pathology, Sapporo Medical University School of Medicine, Japan.
Kojima
T
T
Norioka
K
K
Mochizuki
Y
Y
D028341:Activins; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D007265:Inhibins / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D007375:Interleukin-1; D015850:Interleukin-6; D016212:Transforming Growth Factor beta; D028341:Activins; D007265:Inhibins