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To establish no matter whether CP466722 could inhibit ATM kinase activity in cells and to Aurora B inhibitor decide an efficient concentration for inhibition, HeLa cells were exposed to IR while in the presence of varying concentrations on the inhibitor and phosphorylation of ATM targets was assessed.

Disruption of ATM dependent phosphorylation events as well as inhibition of ATM dependent p53 induction were also observed in MCF 7 human breast cancer cells and main and immortalized diploid human fibroblasts. General, the response to IR in cells handled with CP466722 was much like that witnessed in cells lacking ATM. Because one long term goal is usually to characterize the capability Aurora B inhibitor of CP466722 to sensitize tumors to radiation or chemotherapeutic agents in murine models in vivo, it was important to know if CP466722 was effective at inhibiting Atm kinase in mouse cells. The ATM signaling pathway is conserved from human to mouse and ATM kinase activity can be monitored by analyzing similar downstream events. An exception is phosphorylation of Chk2 on threonine 68 which is difficult to detect in mouse cells.

While ATM is preferentially activated by DSBs and phosphorylates Chk2 on threonine 68, ATR is preferentially activated by stalled replication forks and phosphorylates serine 345 of Chk1. Though CP466722 BI-1356 did not affect ATR kinase activity in vitro, we examined the ability of the compound to affect ATR kinase activity in cells. hTERT immortalized human fibroblasts were treated for 1h with the replication inhibitor aphidicolin in the presence or absence of CP466722. ATR dependent phosphorylation of Chk1 was not inhibited by CP466722, even though ATM dependent phosphorylation of Chk2 was blocked in these cells. Failure to inhibit aphidicolin induced Chk1 phosphorylation in cells lacking ATM provided even more definitive evidence that CP466722 does not inhibit ATR kinase in cells.

Serum starvation resulted in an almost complete loss of AKT phosphorylation.