Phosphorylation of Tip60 by GSK-3 determines the induction of PUMA and apoptosis by p53.

Activation of p53 by DNA damage results in either cell-cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here, we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the proapoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. We demonstrate that GSK-3 phosphorylates serine 86 of the p53-acetyltransferase Tip60. A Tip60(S86A) mutant was less active to induce p53 K120 acetylation, histone 4 acetylation, and expression of PUMA. Our data suggest that GSK-3 mediated Tip60S86 phosphorylation provides a link between PI3K signaling and the choice for or against apoptosis induction by p53.

Nucleocytoplasmic shuttling by nucleoporins Nup153 and Nup214 and CRM1-dependent nuclear export control the subcellular distribution of latent Stat1.

Interferon stimulation of cells leads to the tyrosine phosphorylation of latent Stat1 and subsequent transient accumulation in the nucleus that requires canonical transport factors. However, the mechanisms that control the predominantly cytoplasmic localization in unstimulated cells have not been resolved. We uncovered that constitutive energy- and transport factor-independent nucleocytoplasmic shuttling is a property of unphosphorylated Stat1, Stat3, and Stat5. The NH(2)- and COOH-terminal Stat domains are generally dispensable, whereas alkylation of a single cysteine residue blocked cytokine-independent nuclear translocation and thus implicated the linker domain into the cycling of Stat1. It is revealed that constitutive nucleocytoplasmic shuttling of Stat1 is mediated by direct interactions with the FG repeat regions of nucleoporin 153 and nucleoporin 214 of the nuclear pore. Concurrent active nuclear export by CRM1 created a nucleocytoplasmic Stat1 concentration gradient that is significantly reduced by the blocking of energy-requiring translocation mechanisms or the specific inactivation of CRM1. Thus, we propose that two independent translocation pathways cooperate to determine the steady-state distribution of Stat1.

The novel Bcl-2 inhibitor ABT-737 is more effective in hypoxia and is able to reverse hypoxia-induced drug resistance in neuroblastoma cells.

Neuroblastoma is a common solid tumor of childhood and advanced disease carries a poor prognosis despite intensive multimodality therapy. Hypoxia is a common feature of solid tumors because of poorly organized tumor-induced neovasculature. Hypoxia is associated with advanced stage and poor outcome in a range of tumor types, and leads to resistance to clinically relevant cytotoxic agents in neuroblastoma and other pediatric tumors in vitro. Resistance to apoptosis is a common feature of tumor cells and leads to pleiotropic drug resistance, mediated by Bcl-2 family proteins. ABT-737 is a novel small-molecule inhibitor of Bcl-2 and Bcl-x(L) that is able to induce apoptosis in a range of tumor types. Neuroblastoma cell lines are relatively resistant to ABT-737-induced apoptosis in normoxia, but in contrast to the situation with conventional cytotoxic agents are more sensitive in hypoxia. This sensitization is because of an increase in ABT-737-induced apoptosis and is variably dependent upon the presence of functional hypoxia-inducible factor 1 (HIF-1) α. In contrast to the situation in colon carcinoma and non-small cell lung cancer cells, hypoxia does not result in downregulation of the known ABT-737 resistance factor, Mcl-1, nor any other Bcl-2 family proteins. ABT-737 sensitizes neuroblastoma cells to clinically relevant cytotoxic agents under normal levels of oxygen, and importantly, this sensitization is maintained under hypoxia when neuroblastoma cells are resistant to these agents. Thus rational combinations of ABT-737 and conventional cytotoxics offer a novel approach to overcoming hypoxia-induced drug resistance in neuroblastoma.

Hypoxic human cancer cells are sensitized to BH-3 mimetic­induced apoptosis via downregulation of the Bcl-2 protein Mcl-1.

Solid tumors contain hypoxic regions in which cancer cells are often resistant to chemotherapy-induced apoptotic cell death. Therapeutic strategies that specifically target hypoxic cells and promote apoptosis are particularly appealing, as few normal tissues experience hypoxia. We have found that the compound ABT-737, a Bcl-2 homology domain 3 (BH-3) mimetic, promotes apoptotic cell death in human colorectal carcinoma and small cell lung cancer cell lines exposed to hypoxia. This hypoxic induction of apoptosis was mediated through downregulation of myeloid cell leukemia sequence 1 (Mcl-1), a Bcl-2 family protein that serves as a biomarker for ABT-737 resistance. Downregulation of Mcl-1 in hypoxia was independent of hypoxia-inducible factor 1 (HIF-1) activity and was consistent with decreased global protein translation. In addition, ABT-737 induced apoptosis deep within tumor spheroids, consistent with an optimal hypoxic oxygen tension being necessary to promote ABT-737­induced cell death. Tumor xenografts in ABT-737­treated mice also displayed significantly more apoptotic cells within hypoxic regions relative to normoxic regions. Synergies between ABT-737 and other cytotoxic drugs were maintained in hypoxia, suggesting that this drug may be useful in combination with chemotherapeutic agents. Taken together, these findings suggest that Mcl-1­sparing BH-3 mimetics may induce apoptosis in hypoxic tumor cells that are resistant to other chemotherapeutic agents and may have a role in combinatorial chemotherapeutic regimens for treatment of solid tumors.