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

Abstract

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.

Impaired Nuclear Accumulation and Shortened Phosphorylation of ERK After Growth Factor Stimulation in Cultured Hepatocytes From Rats Exposed to 2-Acetylaminofluorene

“…Preneoplastic lesions, as a result of altered xenobiotic metabolism, appear to be protected from this mitoinhibitory effect [8]. In a previous study, we found that mitoinhibition of AAF-exposed cultured hepatocytes was associated with increased p53 protein expression and reduced pRb hyperphosphorylation but not with p21/CIP1 induction [6]. Also, during in vivo regeneration of AAF-exposed rat liver, the binding of p21/CIP1 to cyclin E or cdk2 was not increased, although cdk2 complex formation and activation were inhibited.…”

“…A sustained ERK activation is required for the induction of cyclin D1, one of the ®rst events of cell-cycle progression [19]. Indeed, our previous studies have demonstrated that mitoinhibition of AAF-exposed primary hepatocytes is associated with a profound inhibition of cyclin D1 induction [6]. Thus, the demonstrated effects of AAF on ERK functions may have severe effects on G 1 progression, contributing to the mitoinhibitory effects of this carcinogen.…”

“…Because the DEN-initiated cells are resistant to this effect, they proliferate selectively. The mitoinhibition of normal hepatocytes occurs before S-phase entry, as shown by the low thymidine labeling index [5], inhibition of pRb hyperphosphorylation, and reduced activation of cdk4 and cdk2 [6]. The promoting capacities of AAF have been attributed to DNA damage, resulting in p53-mediated growth arrest [7,8].…”

Molecular mechanism of G1 arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 inhibitor, in leukemia cells

“…Both CDK4 and 6 are important for cell cycle regulation, specifically G 1 phase progression, with their activity strictly restricted to the G 1 -S phase [3–5]. Mutations in these genes have been found to be significantly associated with tumorigenesis of several cancers [6, 7].…”