it was present read FAQ in both the cytosolic and the mitochondrial fractions, and primarily unphosphorylated on Ser15. Cytosolic accumulation of p53 has been reported to directly activate Bax to insert and oligomerize into the mitochondrial membrane, thus leading to the cascade of mitochondria mediated apoptosis and caspase 3 activa tion. We therefore investigated whether intrinsically generated high cytosolic p53 levels in DRB treated cells were coupled to activation of Bax. We monitored Bax dis tribution by immunofluorescence and confocal micros copy. In untreated cells Bax appeared in a basal cytosolic distribution, whereas 6 hr after DRB treat ment a dot like pattern was displayed by about 20% of cells and then by virtually 100% after 15 hr.

To better evaluate Bax activation, we studied the distribution of endogenous Bax with respect to its mitochondrial localization by fractionation experi ments. Results demonstrated that in untreated cells Bax was free in the cytosol, whereas during DRB treatment it progressively translocated to the mithocondria, where it was retained throughout the entire time course. damage independent andis replication independent, DNA To further investigate the role of mitochondrial p53 in DRB induced apoptosis we used pharmacological manip ulations to distinguish nuclear and mitochondrial p53 functions. T cells were preincubated with two inhibitors of p53 that have different mechanisms of action pifithrin reversibly blocks the p53 mediated transactiva tion of p53 dependent responsive genes, pifithrin blocks the interaction of p53 with Bcl xL and Bcl 2 and selectively inhibits p53 translocation to the mito chondria, without affecting the transactivation function of p53 at the level of the genome.

While the viability level of DRB treated T cells did not change in the presence of PFT, preincubation with PFT conferred partial resistance toward DRB induced death, with viability at 24 hours of 70%. To assess whether the blockage of mitochondrial p53 does increase viability through apoptosis inhibition we analysed caspase 3 acti vation in control T cells treated with DRB, either alone or in the presence of the two p53 inhibitors. We found that p53 dependent apoptosis induced by DNA intercalating agents like Actinomycin D and display a clear p53 accumulation defect following genotoxic stimuli such as ionizing radiation.

However, DRB induced p53 accumulation was comparable to Dacomitinib that of nor mal cells, as assessed by both western blot analysis and confocal microscopy. As in normal T lymphocytes, accumulated p53 was essentially cytosolic, and assessment of their Bax distribution revealed a dot like pattern after DRB treatment, at levels comparable to the control cells. Accordingly, AT and NBS T lymphocytes readily died after DRB treat ment, with caspase 3 activation comparable to that of control T lymphocytes. These results suggest that DRB induced accumulation of p53 and subsequent apoptosis are ATM and NBS1 independent.