Comparable results were obtained with quiescent cultured T lymphocytes. To verify that DRB induced selleck bio apoptosis was not attributable to induction of DNA damage, we assessed phosphoryla tion of the histone variant H2AX on serine 139, an early and specific indicator of DNA double strand breaks, by immunofluorescence and confocal microscopy. As apoptosis itself results in oligonucleo somal fragmentation of DNA and H2AX phosphorylation, these experiments were done in the presence of the caspase inhibitor ZVAD FMK. Untreated cells displayed virtually no H2AX foci, after DRB treatment H2AX foci formation did not occur. We then investigated the molecular pathway elicited by DRB in T lymphocytes. As shown in figure 2C, DRB rap idly stabilized p53, while phosphorylation of p53 at Ser15 was only detectable when p53 accumulation had already reached the high level.

As phosphorylation of Ser15 on p53 is synonymous with the activation of DNA damage dependent pathways in response to cellular insults, this provides further proof that no genotoxic stress is imposed on cells treated with DRB. p53 accumulation was not contrasted by Mdm2, a p53 target required for p53 degradation, as this protein was rapidly down regu lated, possibly due to the known transcriptional inhibi tory effect of DRB. Overall, these results indicate that the stress response elic ited by DRB in human T cells results in replication and DNA damage independent apoptosis. They also suggest that the death pathway thus induced is p53 mediated.

Cytosolic p53 accumulates in pre apoptotic DRB treated T lymphocytes and correlates with Bax activation In the light of DRBs potent inhibition of transcription, we reasoned that the apoptotic pathway it induced did not rely on the transactivation activity of p53. Recent studies have linked the non transcriptional pro apoptotic activity of p53 to its cytosolic or mitochondrial localization. To determine whether this activity contributes to DRB induced apoptosis in human T cells, we monitored the p53 location of DRB treated cells. We first analyzed p53 distribution by immunostaining and confocal microscopy. A time course experiment shown that active caspase 3 becomes detectable 9 hr after DRB treatment, indicating that the signals which initialize the apoptotic cascade must occur earlier.

We therefore performed p53 confocal microscopy analy sis of T lymphocytes harvested at 6 hr from treatment and evaluated its nuclear versus cytosolic localization. p53 was preferentially accumulated in the cytosol. We then prepared cytosolic and mitochondrial fractions of T lymphocytes harvested at 1 3 6 hr from treatment, and determined the distribution of p53 with respect to its mitochondrial location. Purity of the fractions was checked by probing the blots for vinculin and TOM40 Carfilzomib as markers for the cytosolic and mitochondrial compart ments, respectively.