Alteration of G1 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 carcinogenesis models. In the resistant hepatocyte model, AAF is combined with a growth stimulus for efficient promotion of preneoplastic lesions. The promoting property of AAF in this model is closely associated with mito-inhibition 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-genotoxic effects have been implicated. To elucidate the mechanisms of AAF-induced mito-inhibition, we studied the expression of the tumor suppressor protein p53 and the cyclin-dependent kinase (cdk) complexes mediating G1 progression 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 cells. The retinoblastoma protein did not become hyperphosphorylated in AAF-exposed cells. Thus, inhibition of G1 cyclin-cdk activity was implied as a cause of growth arrest. Indeed, G1 cell-cycle arrest was accompanied by reduced induction and nuclear accumulation of the cyclin D1-cdk4 complex and inhibited nuclear translocation of cdk2. Furthermore, the growth arrest was not mediated through p21/waf1 upregulation, although nuclear levels of p53 were increased. Thus, carcinogen-induced mito-inhibition may be effected by altered levels and localization of G1 cyclin-cdk complexes, independent of the upregulation of cdk inhibitory proteins.
Institute of Pathology, The National Hospital, University of Oslo, Norway.
B
B
Lindeman
E
E
Skarpen
G H
GH
Thoresen
T
T
Christoffersen
L
L
Wierød
I H
IH
Madshus
H S
HS
Huitfeldt
D015073:2-Acetylaminofluorene / Q000494:pharmacology*; D000818:Animals; D002273:Carcinogens / Q000494:pharmacology*; D002453:Cell Cycle / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis; D004791:Enzyme Inhibitors; D016193:G1 Phase; D005786:Gene Expression Regulation / Q000187:drug effects*; D016158:Genes, p53; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051381:Rats; D011916:Rats, Inbred F344; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis
D002273:Carcinogens; C493958:Cdkn1a protein, rat; D016895:Culture Media, Serum-Free; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D016159:Tumor Suppressor Protein p53; D004247:DNA; D015073:2-Acetylaminofluorene; D013936:Thymidine