AE cells have an activated p53 pathway, increased apoptosis, and increased sensitivity to DNA damage. (A) Western blot analysis showing higher levels of p53 and p21 proteins in 3 pairs of AE and MIT cultures at week 5 after transduction. (B) RQ-PCR analysis shows increased expression levels of p53 target genes DAPK1 and TP53I3. Three pairs of AE and MIT cultures were analyzed at weeks 1 and 5 after transduction. (C) BrdU staining of AE and MIT cultures shows a decreased percentage of cells in early S phase after irradiation, demonstrating a functional G1 checkpoint. (D) Staining of AE and MIT cells for an early mitotic antigen MPM-2 shows a decreased number of cells in M phase after irradiation, demonstrating a functional G2 checkpoint. Cultures used for experiments shown in panels C,D were 4 weeks old, and flow panels are representative of at least 3 separate stainings. (E) AnnexinV staining reveals increased levels of apoptosis in AE cultures compared with MIT cultures. Six AE and 5 MIT cultures were analyzed at week 5 after transduction. (F) AE and MIT cultures 5 weeks after transduction do not show differences in cell cycle. BrdU was incorporated and cells were stained with Anti-BrdU Ab and 7-AAD. An average and standard deviation from 3 separate experiments are shown. (G-I) AE cells show increased sensitivity to ionizing irradiation, mitomycin C (MMC), and AraC compared with MIT cells. Proliferation assays were performed on paired cultures at 5 weeks after transduction, 72 hours after irradiation, or after 72 hours of incubation with MMC or AraC.
