Phosphorylation at these sites has been demonstrated to regulate the apoptotic activity of p53. Phosphorylation of p53 at serine 15, which has been demonstrated to increase protein stability and activity, may partially account for the increased p53 e pression observed in response to eIF5A1. ERK1 2 and p38 MAPK have both been reported to phosphorylate p53 at several residues, including serine 15. Accordingly, we e amined the effects of chemical inhibitors of p38 MAPK, JNK, and ERK on p53 phosphorylation. Although inhibitors of p38 and JNK did not affect phos phorylation of p53 in response to Ad eIF5A1, the MEK inhibitor, U1026, dramatically reduced phosphorylation at all three sites. The total e pression of p53 was also some what reduced in U1026 treated cells, suggesting that phos phorylation was contributing to stability of the protein.

Transcriptional regulation of pro apoptotic members of the Bcl 2 family is involved in the initiation of apoptosis that is central to the tumor suppressor ac tivity of p53. Increased e pression of the pro apoptotic Bcl 2 family members Ba and Bid, but not Bim, was observed following Ad eIF5A1 infection, suggesting that p53 mediated induction of Bcl 2 pro apoptotic family members may contribute to eIF5A1 induced apoptosis. Quantitative reverse transcription PCR amplification of tumor necrosis factor receptor 1, a p53 transcriptional target, revealed that Ad eIF5A1 infection resulted in increased tran scriptional activity of p53. E pression levels of both TNFR1 and p53 mRNA increased in response to Ad eIF5A1 infection and this up regulation was inhibited by both U1026 and pifithrin, an inhibitor of p53 activity.

This indicates that over e pression of unhypusinated eIF5A1 resulted in increased p53 tran scriptional activity that is at least partially dependent on MEK activity. Inhibitors of p38 MAPK and JNK protect A549 cells from Ad eIF5A1 induced apoptosis ERK, p38, and JNK signaling pathways are involved in both apoptosis and cell growth, depending on the cell type and stimulus. The dependence of eIF5A1 on activa tion of p38, JNK and ERK for induction of apoptosis was evaluated by pre treating A549 cells with specific inhibitors to these kinases and then inducing apoptosis by infecting the cells with Ad eIF5A1.

Since Ad Batimastat eIF5A1 infection is associated with increased e pression and activity of p53, cells were also pre treated with pifithrin in order to deter mine whether eIF5A1 induced apoptosis is dependent on p53 activity in A549 cells. MEK inhibition did not significantly affect induction of apoptosis by Ad eIF5A1. Inhibition of p38 and JNK both significantly reduced eIF5A1 induced apoptosis while use of both inhibitors in combination inhibited apoptosis by appro imately 50%, suggesting that activation of p38 and JNK are both important in the induction of apoptosis by eIF5A1.