RESUMO
BACKGROUND/AIMS: The synthetic estrogen 17alpha-ethinyl estradiol (EE), a potent tumor promoter in rat liver, stimulates growth during short-term treatment but inhibits hepatocyte proliferation upon prolonged treatment. To identify the molecular targets of the mitoinhibitory effect of EE, the expression of proteins regulating G(1)- and S-progression were analyzed during the first cell cycle in EE-treated female Wistar rats. METHODS: Long-term (60 days) EE treatment. Immunohistochemical staining for proliferation cell nuclear antigen (PCNA) to detect cells in S phase and quantification of mitosis. Western blot to monitor protein expression. Cdk2 kinase assay to examine histone H1 phosphorylation. RESULTS: EE reduced the number of cells in S phase and mitosis by about 70%. Cyclin D1 and D3 were unaffected, while cdk4 was moderately decreased. Cyclin E and cdk2 were markedly decreased with concomitant marked reduction of cdk2 kinase activity. EE also decreased cyclin A and increased G1 levels of p53 and p21. CONCLUSIONS: EE causes a cell cycle block before S-phase. The reduction of the cdk2 kinase activity, essential for G1/S-transition, might be involved in the cell cycle block. Also, EE treatment results in p53 activation and upregulation of the cdk inhibitor p21 that might contribute to the G1 arrest.
Assuntos
Quinases relacionadas a CDC2 e CDC28/genética , Ciclina E/genética , Regeneração Hepática/fisiologia , Fígado/fisiologia , Noretinodrel/análogos & derivados , Fase S/fisiologia , Animais , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclina D1/genética , Ciclina D3 , Quinase 2 Dependente de Ciclina , Ciclinas/genética , Replicação do DNA , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Regeneração Hepática/efeitos dos fármacos , Mitose/efeitos dos fármacos , Noretinodrel/uso terapêutico , Ratos , Ratos Wistar , Fase S/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: Examine the mitoinhibitory effect of the liver tumor promoter 2-acetylaminofluorene (2-AAF) in vivo, with focus on the proteins regulating G1- and S progression. METHODS: cdk 2 kinase assay to examine histone H1 phosphorylation. cdk 4 kinase assay to examine whether active cdk 4/cyclin D complexes, capable of phosphorylating Rb, are formed. Western blot to monitor protein expression. RESULTS: cdk 4 kinase-mediated Rb phosphorylation was increased by AAF treatment. Nuclear expression of the transcription factors E2F-1 and E2F-3 was downregulated, while E2F-4 was decreased. 2-AAF treatment also markedly reduced cdk 2 kinase activity/histone H1 phosphorylation during G1/S-transition. Western blot showed loss of nuclear as well as cytoplasmic cyclin A and cyclin B protein after 2-AAF treatment, while the Rb protein level was markedly increased during G1. The cell cycle dependent elevation of nuclear p107 protein, seen in control livers, was not observed in AAF-treated animals. CONCLUSIONS: Effects of 2-AAF; Very low cdk 2 kinase activity that could possibly block G1/S-transition; increased pRb level together with diminished levels of transcription factors E2F-1 and -3, that could be responsible for reducing the expression of E2F target genes such as cyclin A and E2F-1.
