Figure 1.
Expression of non-degradable cyclin B1 leads to a modest increase in mitotic arrest and activation of Cdk1, and rapid apoptosis.
HeLa cells were transfected with 2 µg plasmid DNA encoding GFP vector or encoding non-degradable cyclin B1-R42A-GFP for the times indicated and subjected to: A. Immunoblotting for GFP and cyclin B1. GAPDH was used as a loading control. Apparent molecular weights of the proteins are indicated on the left. B. Cell cycle analysis by propidium iodide staining and flow cytometry. Mitotic arrest is indicated by an increase in the percentage of cells with 4N DNA content. C. Cdk1 assay performed as described in Materials and Methods. 32P incorporation was determined by scintillation counting. Values shown have been corrected for background kinase activity (no substrate) and represent the mean ± standard deviation (n = 3). D. Cell death ELISA, as described in Materials and Methods. Untransfected HeLa cells were used as a negative control. Results given are mean ± standard deviation (n = 3).
Figure 4.
Dose-dependent effects of cyclin B1-R42A-GFP expression on extent of mitotic arrest and cell death.
HeLa cells were transfected with increasing concentrations (0.2–2 µg) of plasmid encoding cyclin B1-R42A-GFP or 2 µg plasmid encoding GFP vector, as indicated, for 24 h and then subjected to propidium iodide staining and analyzed by flow cytometry. The proportion of GFP positive cells was used to assess transfection efficiency. The DNA content of GFP positive cells was used to determine the percentage of transfected cells undergoing mitotic arrest (4N DNA) or apoptosis (sub-G1 DNA). Data shown are mean ± standard deviation of three independent experiments.
Figure 2.
Cyclin B1 overexpression fails to recapitulate effects of vinblastine on phosphorylation of anti-apoptotic Bcl-2 proteins.
HeLa cells were transfected with 2 µg plasmids encoding either wild-type cyclin B1-GFP (Wt), non-degradable cyclin B1-R42A-GFP, or GFP vector for 12–24 h. Untreated untransfected or vinblastine treated (30 nM, 24 h) HeLa cells are shown in the right two lanes. Extracts were prepared and subjected to immunoblotting for the proteins indicated. GAPDH was used as a loading control. Apparent molecular weights (in kDa) are indicated.
Figure 3.
Overexpression of cyclin B1 in synchronized cells induces cell death in G1 phase independent of mitotic arrest.
HeLa cells were synchronized at the G1/S boundary using double thymidine block, released into fresh media, and 1 h post-release were untreated (vehicle), treated with 30 nM vinblastine, or transfected with 2 µg plasmids encoding either GFP vector, wild-type cyclin B1-GFP or cylcin B1-R42A-GFP. Cells were harvested at intervals 10–24 h post-release and extracts subjected to immunoblotting for the indicated proteins. Top panel, experimental scheme, with corresponding cell cycle phases indicated. Bottom panel, immunoblots.