FIGURE 3.

Cells harboring mutations in Arf/p53 are sensitive to CPT. A, MEFs harboring mutations in Arf and p53 are sensitive to CPT. All experiments were performed as outlined in Fig. 1. Both primary and immortalized Arf and p53 KO MEFs were sensitive to CPT (similar to immortalized WT MEFs). Survival rates were plotted as in Fig. 1A. Representative images of the cell cultures are also shown in supplemental Fig. S1A. B and C, the effects of CPT treatment were examined over time. Both Arf and p53 KO MEFs show signals for γH2AX and cleaved Parp1 after CPT treatment. The experiments were performed as outlined in Fig. 1F. Unlike WT MEFs, both primary and immortalized p53 KO MEFs showed increased levels of H2AX and γH2AX and a cleaved-Parp1 signal, suggesting apoptosis induction. Compared with p53 KO MEFs, Arf KO MEFs showed early onset of cell death (see also supplemental Fig. S1B). Therefore, Arf KO MEFs were subsequently treated with 10 nm CPT. D, cell cycle arrest was examined by FACS. Arf KO MEFs arrested in G₂ phase, whereas p53 KO MEFs showed an 8N chromosome peak, which often indicates cell death because of mitotic catastrophe. E, model showing the cellular response to CPT. While normal cells reduce their expression of H2AX and become quiescent (with impaired checkpoint responses), immortalized cells expressing γH2AX are killed preferentially.