Mitoinhibitory effects of the tumor promoter 2-acetylaminofluorene in rat liver: loss of E2F-1 and E2F-3 expression and cdk 2 kinase activity in late G1.
OBJECTIVE
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.
Ohlson
Lena C E
LC
Natural Science Section, Södertörns högskola (University College), P.O. Box 4101, 141 89 Huddinge, Sweden.
Koroxenidou
Lena
L
Porsch-Hällström
Inger
I
D015073:2-Acetylaminofluorene / Q000633:toxicity*; D000818:Animals; D042846:CDC2-CDC28 Kinases / Q000235:genetics*; D002273:Carcinogens / Q000633:toxicity; D018797:Cell Cycle Proteins / Q000235:genetics*; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000187:drug effects / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D050699:E2F3 Transcription Factor; D016193:G1 Phase; D005786:Gene Expression Regulation / Q000187:drug effects*; D006657:Histones / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D008297:Male; D009687:Nuclear Proteins / Q000187:drug effects / Q000235:genetics; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D014157:Transcription Factors / Q000235:genetics*
D002273:Carcinogens; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D050699:E2F3 Transcription Factor; C493755:E2f1 protein, rat; D006657:Histones; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D015073:2-Acetylaminofluorene; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; C495903:Cdk4 protein, rat; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases