The effects of dietary 2-acetylaminofluorene (2-AAF) on cell cycle-related proteins was studied in regenerating livers from male Wistar rats. The levels of cyclins, cyclin dependent kinases (cdks), and related proteins were studied at different times during the first cell cycle after partial hepatectomy (PH). The frequency of proliferation cell nuclear antigen (PCNA)-positive nuclei, a marker of S phase progression, was almost zero during the first 27 hours after PH in the mitoinhibited 2-AAF-treated rats, while about 50% of the nuclei were labeled 24 hours after PH in control animals. Accordingly, Western blot tests showed markedly elevated PCNA protein levels from 18 hours to the end of S phase in untreated animals but no upregulation in response to 2-AAF. Compared with control animals, animals treated with 2-AAF showed increased levels of cdk 4 and cyclin D 3 from 12 and 15 hours after PH, respectively, and altered cyclicity in cyclin D 3 expression. No effects on cyclin E were observed, while the increase in cdk 2 levels in control animals during late G 1 /S (15-27 hours) was abolished by 2-AAF. p53 was induced by 2-AAF treatment during the same period, with a peak at 24 hours. The protein detected with p21 antibodies was highly expressed in unstimulated hepatocytes in control animals, and further increased by 2-AAF. The expression was sustained until 15 hours after PH in control rats while 2-AAF-treated animals lacked detectable protein during this period; however, a transient increase was observed at 21 hours. Thus, 2-AAF affects several parameters of cell cycle regulation of possible relevance for its inhibitory effects on hepatocyte proliferation in vivo. (HEPATOLOGY 1998;27:691-696.)Many liver tumor promoters such as 2-acetylaminofluorene (2-AAF), phenobarbital, orotic acid, clofibrate and ethinylestradiol have inhibitory effects on proliferation of hepatocytes in vivo during long-term treatment, 1-5 which is believed to be an important aspect of the promotion mechanism. In the classical resistant hepatocyte model (RH-model) for rat liver carcinogenesis, where promotion is performed by dietary 2-AAF treatment combined with a partial hepatectomy (PH), the selective growth advantage of preneoplastic foci during treatment has been suggested to be caused by the inhibited proliferation of hepatocytes surrounding the lesions, combined with focal resistance to 2-AAF-induced inhibition. 1 This makes the putatively preneoplastic foci, in contrast to the surrounding tissue, able to respond to the proliferative stimulus provided by the PH. The mitoinhibitory effects of 2-AAF are caused by the formation of sulfated metabolites of N-hydroxyacetylaminofluorene, 6 which is low in the lesions compared with the surrounding hepatocytes. 7 These sulfated metabolites give rise to two different Nacetylated adducts causing major distortion of the DNA structure, and lead to an efficient DNA replication block both in vitro and in vivo. 6,[8][9] The molecular mediators of the inhibitory signals generated by 2-AAF are not known....
