To find out if selleckchem Alisertib a different p53 status of colon carcinoma cells influences the TNF��-mediated apoptosis through the p-p38/DAPK axis we performed some key experiments in HCT116 p53?/? cells and in HT29 (p53 mutant) cells. Interestingly, HCT116 p53 deficient tumor cells and HT29 mutant colorectal cancer cells subjected to TNF�� showed slight differences in cell death induction. Whereas in both HCT116 deficient cells and HCT116 wild-type cells apoptosis induction started already after 24 hours (but to a lower extent in the HCT116 deficient cells with 13.3% versus 8.2% at 24 hours, 19.2% versus 9.4% at 48 hours, respectively) the mutant HT29 cells displayed a cell death delay starting initially after 48 hours but reaching only the levels of HCT116 deficient cells (Figure 6A, and Figure 7A).

Interestingly, in mutant HT29 cells p-p38 induction was highest at 24 hours and correspondingly the DAPK protein amounts increased at 48 hours and later. In HCT p53?/? cells p-p38, as well as DAPK levels started increasing at earlier time points comparable with p53 wild-type cells (Figures 6B and 7B). Figure 6 TNF�� induced DAPK-mediated apoptosis and DAPK/p38 co-localization in HCT116 p53 deficient cells. A: Annexin-V measurements of control HCT116 p53?/? cells (ctrl) and HCT116 p53?/? cells subjected to TNF��. … Figure 7 TNF�� induced DAPK-mediated apoptosis and DAPK/p38 co-localization in HT29 p53 mutant cells. A: Annexin-V measurements of control HT29 cells (ctrl) and HT29 cells subjected to TNF��. B: Lysates of HT29 cells subjected to TNF�� were …

Furthermore, they showed a similar pattern of increase in p-p38 (HT29: fourfold after 6 hours and sixfold after 24 hours with a significant decrease at later time points; HCT p53?/?: fourfold after 6 hours and complete loss at later time points. Whereas there was a similar and continuous increase in DAPK protein levels at 48 hours and later in both p53 inactive cell lines (Figures 6B and 7B), the inactive pDAPK308 protein level simultaneously decreased, starting earlier in HCT p53?/? cells than in HT29 mutant cells. In p53?/? cells the ratio of pDAPK308/DAPK decreased 4.3-fold early at 6 hours and in HT29 cells the ratio of pDAPK308/DAPK started decreasing later with 1.6-fold at 24 hours. In general, pDAPK308 nearly disappeared at 48 hours and at later time points in all three colorectal cancer cell lines (Figures 3C, ,6B,6B, and 7B).

The time delay observed between p53?/? cells and HT29 cells is well in line with the observed pattern of apoptosis induA 49-year-old man came to the emergency center presenting with epigastric pain and melena in April 2002. Esophagogastroduodenoscopy showed a centrally ulcerative huge mass with bleeding in the superior wall of the duodenal 1st Cilengitide portion. A biopsy was performed, and the mass was confirmed as a spindle cell type GIST with strong c-kit immunoreactivity. A CT scan revealed a 2.