2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent release from contact inhibition in WB-F344 cells: involvement of cyclin A.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent tumor promoter ever tested in rodents. Although it is known that most of TCDD actions are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact inhibition is one characteristic hallmark in tumorigenesis. In rat liver epithelial WB-F344 cells, TCDD induces a release from contact inhibition, which is manifested by a twofold increase in cell number when TCDD (1 nM for 48 h) is added to confluent cells in the presence of serum, but not when given to exponentially growing or subconfluent, serum-deprived WB-F344 cells. Loss of G1 arrest was also shown by flow cytometric analysis. We demonstrate that TCDD treatment significantly increases cyclin D2 and cyclin A protein levels and show by immunofluorescence that these proteins accumulate in the nucleus. Although TCDD treatment leads to a strong increase in cyclin D2/cdk4 and cyclin A/cdk2 complex formation, we could only detect an elevation of cyclin A/cdk2 activity. In accordance with a lack of elevated cdk4 activity, no decrease in the amount of hypophosphorylated retinoblastoma protein could be shown after TCDD treatment. The importance of increased cyclin A/cdk2 activity for TCDD-dependent release from contact inhibition was shown by the fact that the cdk2/cdc2-specific inhibitor olomoucine (25 microM) abolished TCDD response. These data indicate cyclin A-dependent loss of G1 arrest after TCDD treatment mainly downstream of the retinoblastoma protein.
Dietrich
C
C
Institute of Toxicology, Johannes Gutenberg-University, Obere Zahlbacherstrasse 67, 55131 Mainz, Germany. cdietric@mail.uni-mainz.de
Faust
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D
Budt
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Moskwa
M
M
Kunz
A
A
Bock
K-W
KW
Oesch
F
F
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002273:Carcinogens / Q000494:pharmacology*; D002460:Cell Line; D019925:Cyclin A / Q000378:metabolism / Q000502:physiology*; D056742:Cyclin D2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007701:Kinetin; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D011687:Purines / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D016160:Retinoblastoma Protein / Q000378:metabolism
D002273:Carcinogens; C486197:Ccnd2 protein, rat; D019925:Cyclin A; D056742:Cyclin D2; D016213:Cyclins; D004791:Enzyme Inhibitors; D000072317:Polychlorinated Dibenzodioxins; D011687:Purines; D016160:Retinoblastoma Protein; C090046:olomoucine; D018844:Cyclin-Dependent Kinases; D007701:Kinetin