FIG. 5.
Deregulated G2/M-phase transition in p53-deficient cells. (A) Nuclear localization of cyclin B1 in wild-type cells (W.T.; IMR-90) and fibroblasts expressing low levels of p21 owing to expression of HPV16 E6 (+E6) as described in Materials and Methods. Formalin-fixed cells were stained with cyclin B1- (fluorescein; a and d) and p21Cip1-specific (Texas red; b and e) antibodies. Nuclei were counterstained with Hoechst 33258 to assess the state of DNA condensation (c and f). Experimental conditions were the same as those described for Fig. 1. Note the absence of nucleoli and the signs of DNA condensation in E6 cells accumulating nuclear cyclin B1. Bar, 10 μm. (B) Cyclin B1- and Cdk2-associated histone H1 kinase activity from aliquots prepared from synchronized wild-type and E6 fibroblasts. Cells were synchronized at the G1/S-phase boundary by aphidicolin block as described in the legend to Fig. 2. (C) Accelerated entry into mitosis of p53− p21− cells. The late stages of the cell cycle in synchronized wild-type and E6 cultures were analyzed based on subcellular distribution of cyclin B1. Cells were released from G1/S-phase block (hydroxyurea) for 6 and 10 h as described in Materials and Methods. Note that only cells accumulating cytoplasmic and nuclear cyclin B1 were scored. The following cyclin B1-specific staining patterns were distinguished: Cyt, cytoplasmic (like in Fig. 1H); CN, cytoplasmic and nuclear, no visible nucleoli (like in Fig. 1H); CN*, cytoplasmic and nuclear, with visible nucleoli (like in Fig. 1H); Nuc, predominantly nuclear with visible nucleoli (like in Fig. 1H and in 3A); PM, prophase and metaphase (like in Fig. 1F).