Alteration of G(1) cell-cycle protein expression and induction of p53 but not p21/waf1 by the DNA-modifying carcinogen 2-acetylaminofluorene in growth-stimulated hepatocytes in vitro

2-Acetylaminofluorene (AAF) is a potent tumor promoter-in rat liver carcino genesis models. In the resistant hepatocyte model, AAF is combined with a g rowth stimulus for efficient promotion of preneoplastic lesions. The promot ing property of AAF in this model is closely associated with mito-inhibitio n of normal hepatocytes, an effect to which initiated cells are resistant. How AAF induces growth arrest is not known, but genotoxic as well as non-ge notoxic effects have been implicated. To elucidate the mechanisms of AAF-in duced mite-inhibition, we studied the expression of the tumor suppressor pr otein p53 and the cyclin-dependent kinase (cdk) complexes mediating G(1) pr ogression and S-phase entry. Hepatocytes were isolated from male Fisher 344 rats fed either a control diet or a diet supplemented with 0.02% AAF for 1 wk and cultured in a defined serum-free medium containing epidermal growth factor, insulin, and dexamethasone. Thymidine labeling revealed a profound inhibition of DNA synthesis in AAF-exposed cells compared with control cel ls. The retinoblastoma protein did not become hyperphosphorylated in AAF-ex posed cells. Thus, inhibition of G(1) cyclin-cdk activity was implied as a cause of growth arrest. Indeed, G(1) cell-cycle arrest was accompanied by r educed induction and nuclear accumulation of the cyclin D1-cdk4 complex and inhibited nuclear translocation of cdk2. Furthermore, the growth arrest wa s not mediated through p21/waf1 upregulation, although nuclear levels of p5 3 were increased. Thus, carcinogen-induced mite-inhibition may be effected by altered levels and localization of G(1) cyclin-cdk complexes, independen t of the upregulation of cdk inhibitory proteins. (C) 1999 Wiley-Liss, Inc.