Figure 5.

Delayed recovery from cell cycle arrest in pemetrexed-treated UNG^−/− cells. (a) Cell cycle progression was monitored using PI staining and flow cytometry. UNG^+/+ and UNG^−/− cells treated with IC₅₀ level pemetrexed (UNG^+/+, 200 nM; UNG^−/− 25 nM) both show evidence of S-phase accumulation after 25 h of exposure. When allowed to recover in drug-free media, UNG^+/+ cells are capable of resuming cell cycle (return of G1 peak in +48 h sample) while UNG^−/− cells are not. (b) Western blot from UNG^+/+ and UNG^−/− cells treated for 0–72 h with 25 nM pemetrexed showing increased sensitivity in UNG^−/− cells to pemetrexed-induced phosphorylation of chk1 and cdc2, S-phase checkpoint kinases. (c) Cell cycle histograms for cells treated with supplemental thymidine. Cells were treated with 10 μM thymidine for 24 h (No Pem, +Thy), co-treated with IC₅₀ level pemetrexed and thymidine for 24 h (24 h, +Thy) or treated with IC₅₀ level pemetrexed for 24 h and allowed to recover for 48 h in media containing 10 μM thymidine (48 h post, +Thy)