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Acetylation of p53 at carboxyl-terminal lysine residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Here we demonstrate that p53 acetylation at Lys-320/Lys-373/Lys-382 is also required for its transcription-independent functions in BAX activation, reactive oxygen species production, and apoptosis in response to the histone deacetylase inhibitors (HDACi) suberoylanilide hydroxamic acid and LAQ824. Knock-out of p53 markedly reduced HDACi-induced apoptosis. Unexpectedly, expression of transactivation-deficient p53 variants sensitized p53-null cells to HDACi-mediated BAX-dependent apoptosis, whereas knockdown of endogenous mutant p53 in cancer cells reduced HDACi-mediated cytotoxicity. Evaluation of the mechanisms controlling this response led to the discovery of a novel interaction between p53 and Ku70. The association between these two proteins was acetylation-independent, but acetylation of p53 could prevent and disrupt the Ku70-BAX complex and enhance apoptosis. These results suggest a new mechanism of acetylated p53 transcription-independent regulation of apoptosis.

Recent studies have demonstrated that the cell growth and antiapoptotic actions of androgen could be dissociated from the transcriptional activity of the receptor and were, instead, mediated by activation of a mitogen-activated protein kinase pathway. This finding suggests an important cellular function of androgen receptor (AR) outside the nucleus. In this report, we demonstrate that androgen activates phosphatidylinositol 3-kinase (PI3K) and Akt, including AKT1 and AKT2, in AR-positive cells. Androgen-induced cell growth and survival were inhibited by PI3K inhibitor and dominant-negative Akt. AR interacts with the p85alpha regulatory subunit of PI3K, and its binding affinity is increased after androgen stimulation. The sites of interaction on the two proteins were mapped to the C-terminal Src-homology 2 domain of p85alpha and N terminus of AR. Activation of PI3K/Akt by androgen was inhibited by dominant-negative Src. Neither N-terminal truncated nor proline-rich region-deleted AR mutants, which are unable to bind to p85alpha and Src, respectively, was able to mediate androgen-induced PI3K/Akt activation. AR with deletion of C-terminal region including ligand binding domain, however, retains the ability to activate PI3K/Akt upon androgen stimulation, which supports the notion that nongenomic function of androgen is mediated by its interaction with membrane receptors (1, 3, 4). These findings indicate that a triple complex between AR, p85alpha, and Src is required for androgen-stimulated PI3K/Akt activation, and that the PI3K/Akt pathway, in addition to mitogen-activated protein kinase, mediates androgen-induced cell growth and cell survival.

Bcr-Abl, the product of the protooncogene bcr-abl, is a constitutively active protein-tyrosine kinase that is highly expressed in chronic myelogenous leukemia and in acute myeloid leukemia cells. Because Bcr-Abl is known to provide mitogenic signals through suppression of apoptosis, we investigated the effect of this oncogene product on signaling by tumor necrosis factor (TNF), a proapoptotic cytokine. We used a bcr-abl-deficient human megakaryocytic leukemia cell line MO7E and an isogenic MBA cell line stably transfected with bcr-abl. Electrophoretic mobility shift assay revealed that TNF activated the nuclear transcription factor NF-kappaB in MO7E cells but not in MBA cells. The impaired NF-kappaB activation in Bcr-Abl-expressing cells was not due to absence of the NF-kappaB proteins p65, p50, or p100 or of IkappaBalpha or IkappaBbeta. Okadaic acid-induced NF-kappaB activation was unaffected by Bcr-Abl expression. TNF induced IkappaBalpha phosphorylation and degradation in MO7E cells but not in MBA cells. The suppression of TNF-induced NF-kappaB activation by Bcr-Abl was not restricted to MBA cells, because ectopic expression of Bcr-Abl in human acute myeloid leukemia HL-60 cells also blocked TNF-induced NF-kappaB activation. When examined for the TNF receptors by the radioreceptor assay, flow cytometry, or Western blot analysis, we found that Bcr-Abl expression down-regulated the expression of the TNF receptors. The RNase protection assay and Northern blot analysis revealed the transcriptional down-regulation of the TNF receptor by Bcr-Abl protein. Overall, these results indicate that ectopic expression of Bcr-Abl interferes with the TNF signaling pathway through the down-regulation of TNF receptors.