No abstract

The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate crosssensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.

Gastric cancer is highly refractory to DNA-damaging therapies. We therefore studied both gene mutation and protein expression of p53 and Bax in a cohort of 116 patients with gastric cancer who underwent R0-resection with a curative intent. Bax mutation was independent from severe microsatellite instability (MSI), that is, global mismatch repair deficiency as determined by analysis of BAT-25/BAT-26 microsatellite markers. Thus, Bax-frameshift mutation is a feature of tumors with low MSI. In contrast and as expected, no p53 mutations were observed in the microsatellite instable tumors. p53 Mutation or p53 overexpression did not have an impact on disease prognosis. p53-Inactivation was, however, associated with an extremely poor prognosis in the subgroup of patients with Bax-mutated tumors. Thus, we show for the first time that the combined mutation of p53 and Bax, two key regulators of the mitochondrial apoptosis pathway, results in an extremely aggressive tumor biology and poor clinical prognosis.