A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear Among the protein kinases that have been purified from or localized to the nuclei of eucaryotic cells, the two beststudied enzymes, referred to as NI (6,39,41) and NII (6,11,31,40) activated by double-stranded RNA (33) specifically phosphorylates the translation factor EIF-2a (9). The enzyme isolated from mouse spleen nuclei by Ohtsuki et al0was stimulated up to fivefold by DNA specifically for phosphorylation of two small nonhistone proteins (26-28), and histone phosphorylation by the chromatin-associated cGMP-dependent kinase is also stimulated by DNA (12); in both of these cases, however, the stimulation results from interaction of DNA with the substrate rather than with the enzyme (12,26

Metformin has been a frontline therapy for type 2 diabetes (T2D) for many years. Its effectiveness in T2D treatment is mostly attributed to its suppression of hepatic gluconeogenesis; however, the mechanistic aspects of metformin action remain elusive. In addition to its glucose-lowering effect, metformin possesses other pleiotropic health-promoting effects that include reduced cancer risk and tumorigenesis. Metformin inhibits the electron transport chain (ETC) and ATP synthesis; however, recent data reveal that metformin regulates AMP-activated protein kinase (AMPK) and the mechanistic target of rapamycin complex 1 (mTORC1) by multiple, mutually nonexclusive mechanisms that do not necessarily depend on the inhibition of ETC and the cellular ATP level. In this review, we discuss recent advances in elucidating the molecular mechanisms that are relevant for metformin use in cancer treatment.

The transcription factor NF-B is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. However, the signal transduction pathways regulating NF-B activation in human neutrophils in response to stimulation with tumor ne-

Cutaneous T-cell lymphoma (CTCL) is characterized by constitutive activation of nuclear factor kB (NF-kB), which plays a crucial role in the survival of CTCL cells and their resistance to apoptosis. NF-kB activity in CTCL is inhibited by the proteasome inhibitor bortezomib; however, the mechanisms remained unknown. In this study, we investigated mechanisms by which bortezomib suppresses NF-kB activity in CTCL Hut-78 cells. We demonstrate that bortezomib and MG132 suppress NF-kB activity in Hut-78 cells by a novel mechanism that consists of inducing nuclear translocation and accumulation of IkBa (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), which then associates with NF-kB p65 and p50 in the nucleus and inhibits NF-kB DNA binding activity. Surprisingly, however, while expression of NF-kB-dependent antiapoptotic genes cIAP1 and cIAP2 is inhibited by bortezomib, expression of Bcl-2 is not suppressed. Chromatin immunoprecipitation indicated that cIAP1 and cIAP2 promoters are occupied by NF-kB p65/50 heterodimers, whereas Bcl-2 promoter is occupied predominantly by p50/50 homodimers. Collectively, our data reveal a novel mechanism of bortezomib function in CTCL and suggest that the inhibition of NF-kB-dependent gene expression by bortezomib is gene specific and depends on the subunit composition of NF-kB dimers recruited to NF-kB-responsive promoters. Mol Cancer Res; 9(2); 183-94. Ó2011 AACR.

Neutrophils are among the first circulating leukocytes involved in acute inflammatory processes. Transcription factor NF-κB plays a key role in the inflammatory response, regulating the expression of proinflammatory and anti-apoptotic genes. Recently we have shown that human neutrophils contain a significant amount of NF-κB inhibitor, IκBα, in the nucleus of unstimulated cells. The present objective was to examine the mechanisms controlling the nuclear content of IκBα in human neutrophils and to determine whether increased accumulation of IκBα in the nucleus is associated with increased neutrophil apoptosis. We show for the first time that neutrophil stimulation with pro-inflammatory signals results in degradation of IκBα that occurs in both cytoplasm and nucleus. Prolonged (2-h) stimulation with TNF and LPS induces resynthesis of IκBα that is again translocated to the nucleus in human neutrophils, but not in monocytic cells. Leptomycin B, a specific inhibitor of nuclear export, increases nuclear accumulation of IκBα in stimulated neutrophils by blocking the IκBα nuclear export, and this is associated with inhibition of NF-κB activity, induction of caspase-3 activation, and apoptosis. Based on our data we present a new model of NF-κB regulation in human neutrophils by nuclear IκBα. Our results demonstrate that the NF-κB activity in human neutrophils is regulated by mechanisms clearly different from those in monocytes and other human cells and suggest that the increased nuclear content of IκBα in human neutrophils might represent one of the underlying mechanisms for the increased apoptosis in these cells.

Background: IL-8 promotes angiogenesis and metastasis in ovarian cancer. Results: Proteasome inhibition induces specific recruitment of IKK␤, EGR-1, and S536P-p65 to the IL-8 promoter. Conclusion:The increased IKK␤, EGR-1, and S536P-p65 recruitment results in the increased IL-8 expression and release in ovarian cancer cells. Significance: The BZ-increased IL-8 release may be responsible for the BZ-limited effectiveness in ovarian cancer treatment.

Edited by Alex TokerAMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetylCoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects.Acetylation is one of the epigenetic post-translational modifications of histones; it affects chromatin structure and regulates diverse cellular functions, such as gene expression, DNA replication and repair, and cellular proliferation (1, 2). Acetylation and deacetylation of chromatin histones, mediated by histone acetyltransferases (HATs) 3 and histone deacetylases (HDACs), respectively, represent the major mechanisms for epigenetic gene regulation. The dynamic balance between histone acetylation and deacetylation, mediated by the activities of HATs and HDACs, is stringently regulated in healthy cells but is often dysregulated in cancer (3, 4).Histone acetylation depends on intermediary metabolism for supplying acetyl-CoA in the nucleocytosolic compartment (5). In mammalian cells, the nucleocytosolic enzyme ATP-citrate lyase is the major source of acetyl-CoA for histone acetylation (6). Another mechanism for generation of acetyl-CoA in the nucleus involves translocation of pyruvate dehydrogenase from mitochondria to the nucleus (7). In yeast, global histone acetylation depends on nucleocytosolic acetyl-CoA produced by acetyl-CoA synthetase (5). In both yeast and mammalian cells, the nucleocytosolic acetyl-CoA is the link among cellular energy, carbon metabolism, histone acetylation, ...

The advanced stages of cutaneous T cell lymphoma (CTCL) are characterized not only by decreased levels of pro-inflammatory cytokines, resulting in high susceptibility to infections, but also by high constitutive activity of NFκB, which promotes cell survival and resistance to apoptosis. The increased expression of the proto-oncogene Bcl3 belonging to IκB family is associated with the pathogenesis of the different types of human cancer, yet, the function and regulation of Bcl3 in CTCL have not been studied. Here, we show that Bcl3 is highly expressed in CTCL Hut-78 and HH cells. The suppression of Bcl3 levels decreases the expression of the pro-survival genes cIAP1 and cIAP2, reduces cell viability, and increases CTCL apoptosis. Interestingly, Bcl3 suppression concomitantly increases expression and the release of the pro-inflammatory cytokines IL-8 and IL-17 in CTCL cells. Chromatin immunoprecipitation studies show that Bcl3 regulates cIAP1, cIAP2, IL-8 and IL-17 gene expression through direct binding to their promoters. Bcl3 expression is regulated by bortezomib (BZ)-mediated proteasome inhibition, and BZ inhibits Bcl3 recruitment to its target promoters, resulting in decreased expression of cIAP1 and cIAP2, but increased expression of IL-8 and IL-17. The Bcl3 expression is regulated through NFκB subunit exchange on Bcl3 promoter. In untreated cells, the Bcl3 promoter is occupied predominantly by p65/p50 heterodimers, inducing Bcl3 expression; however, in BZ-treated cells, the p65/50 heterodimers are replaced by p52 subunits, resulting in Bcl3 transcriptional repression. These data provide the first insights into the function and regulation of Bcl3 in CTCL, and indicate that Bcl3 has an important pro-survival and immunosuppressive role in these cells.