FIG. 6.

Cells lacking p53 attenuate the DNA damage-induced G₂ checkpoint after multiple population doublings. (A) Asynchronously growing E6 and LXSN cells varying in PDL were continuously exposed to complete medium containing 100 nM ADR or medium alone for 24 h, fixed, and stained with DAPI, and the MI was determined. (B) Late-passage cells were synchronized by density arrest followed by aphidicolin synchronization. At 4 h after release, they were pulsed with 2 μM ADR for 1 h. At 24 h after release from synchronization, when G₂ maintenance would be expected, the medium was replaced with complete medium containing aphidicolin (3 μg/ml) or nocodazole (0.05 μg/ml) for an additional 24 h. Cells were then fixed, stained, and examined by flow cytometry. (C) p21 loss is not involved in the attenuation of the G₂ checkpoint in late-passage E6 cells. Synchronized, ADR-pulsed early-passage E6 and late-passage LXSN and E6 cells were harvested at the indicated time points. p21 levels were evaluated by Western blotting.