Regulation of the mitogen-activated protein kinase kinase (MEK)-1 by NAD(+)-dependent deacetylases.

Sirtuins are class III deacetylases that regulate many essential processes, including cellular stress, genome stability and metabolism. Although these NAD(+)-dependent deacetylases control adaptive cellular responses, identification of sirtuin-regulated signaling targets remain under-studied. Here, we demonstrate that acetylation of the mitogen-activated protein kinase kinase-1 (MEK1) stimulates its kinase activity, and that acetylated MEK1 is under the regulatory control of the sirtuin family members SIRT1 and SIRT2. Treatment of cells with sirtuin inhibitors, or siRNA knockdown of SIRT1 or SIRT2 proteins, increases MEK1 acetylation and subsequent phosphorylation of the extracellular signal-regulated kinase. Generation of an acetyl-specific MEK1 antibody demonstrates that endogenous acetylated MEK1 is extensively enriched in the nucleus following epidermal growth factor  stimulation. An acetyl-mimic of MEK1 increases inappropriate growth properties, suggesting that acetylation of MEK1 has oncogenic potential.

Phosphorylation of RelA/p65 promotes DNMT-1 recruitment to chromatin and represses transcription of the tumor metastasis suppressor gene BRMS1.

The majority of patients with lung cancer present with metastatic disease. Chronic inflammation and subsequent activation of nuclear factor-κB (NF-κB) have been associated with the development of cancers. The RelA/p65 subunit of NF-κB is typically associated with transcriptional activation. In this report we show that RelA/p65 can function as an active transcriptional repressor through enhanced methylation of the BRMS1 (breast cancer metastasis suppressor 1) metastasis suppressor gene promoter via direct recruitment of DNMT-1 (DNA (cytosine-5)-methyltransferase 1) to chromatin in response to tumor necrosis factor (TNF). TNF-mediated phosphorylation of S276 on RelA/p65 is required for RelA/p65-DNMT-1 interactions, chromatin loading of DNMT-1 and subsequent BRMS1 promoter methylation and transcriptional repression. The ability of RelA/p65 to function as an active transcriptional repressor is promoter specific, as the NF-κB-regulated gene cIAP2 (cellular inhibitor of apoptosis 2) is transcriptionally activated whereas BRMS1 is repressed under identical conditions. Small-molecule inhibition of either of the minimal interacting domains between RelA/p65-DNMT-1 and RelA/p65-BRMS1 promoter abrogates BRMS1 methylation and its transcriptional repression. The ability of RelA/p65 to directly recruit DNMT-1 to chromatin, resulting in promoter-specific methylation and transcriptional repression of tumor metastasis suppressor gene BRMS1, highlights a new mechanism through which NF-κB can regulate metastatic disease, and offers a potential target for newer-generation epigenetic oncopharmaceuticals.

Copine-I represses NF-kappaB transcription by endoproteolysis of p65.

Nuclear factor-kappaB (NF-kappaB) is a dynamic transcription factor that regulates important biological processes involved in cancer initiation and progression. Identifying regulators that control the half-life of NF-kappaB is important to understanding molecular processes that control the duration of transcriptional responses. In this study we identify copine-I, a calcium phospholipid-binding protein, as a novel repressor that physically interacts with p65 to inhibit NF-kappaB transcription. Knockdown of copine-I by siRNA increases tumor necrosis factor alpha-stimulated NF-kappaB transcription, while copine-I expression blocks endogenous transcription. Copine-I abolishes NF-kappaB transcription by inducing endoprotease processing of the N-terminus of p65, a process antagonized by IkappaB alpha. Copine-I stimulates endoproteolysis of p65 within a conserved region that is required for base-specific contact with DNA. p65 proteins lacking the N-terminus fail to bind to DNA and act as dominant-negative molecules that inhibit NF-kappaB transcription. Our work provides evidence that copine-I regulates the half-life of NF-kappaB transcriptional responses through a novel mechanism that involves endoproteolysis of the p65 protein.

Nuclear IKK activity leads to dysregulated notch-dependent gene expression in colorectal cancer.

Nuclear functions for IkappaB kinase (IKK), including phosphorylation of histone H3 and nuclear corepressors, have been recently described. Here, we show that IKK is activated in colorectal tumors concomitant with the presence of phosphorylated SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor that is aberrantly localized in the cytoplasm. In these tumors, IKKalpha associates to the chromatin of specific Notch targets, leading to the release of SMRT. Abrogation of IKK activity by BAY11-7082 or by expressing dominant negative IKKalpha restores the association of SMRT with Notch target genes, resulting in specific gene repression. Finally, BAY11-7082 significantly reduces tumor size in colorectal cancer xenografts (CRC-Xs) implanted in nude mice.

The putative oncoprotein Bcl-3 induces cyclin D1 to stimulate G(1) transition.

Bcl-3 is a distinctive member of the IkappaB family of NF-kappaB inhibitors because it can function to coactivate transcription. A potential involvement of Bcl-3 in oncogenesis is highlighted by the fact that it was cloned due to its location at a breakpoint junction in some cases of human B-cell chronic lymphocytic leukemia and that it is highly expressed in human breast tumor tissue. To analyze the effects of Bcl-3 dysregulation in breast epithelial cells, we created stable immortalized human breast epithelial cell lines either expressing Bcl-3 or carrying the corresponding vector control plasmid. Analysis of the Bcl-3-expressing cells suggests that these cells have a shortened G(1) phase of the cell cycle as well as a significant increase in hyperphosphorylation of the retinoblastoma protein. Additionally, the cyclin D1 gene was found to be highly expressed in these cells. Upon further analysis, Bcl-3, acting as a coactivator with NF-kappaB p52 homodimers, was demonstrated to directly activate the cyclin D1 promoter through an NF-kappaB binding site. Therefore, our results demonstrate that dysregulated expression of Bcl-3 potentiates the G(1) transition of the cell cycle by stimulating the transcription of the cyclin D1 gene in human breast epithelial cells.

Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38.

The serine/threonine kinase Akt/PKB is a potent regulator of cell survival and has oncogenic transformation potential. Previously, it has been shown that Akt can activate the transcription factor NF-kappaB and that this functions to block apoptosis induced by certain stimuli. The mechanism whereby Akt activates NF-kappaB has been controversial, with evidence supporting induction of nuclear translocation of NF-kappaB via activation of IkappaB kinase activity and/or the stimulation of the transcription function of NF-kappaB. Here we demonstrate that Akt targets the transactivation function of NF-kappaB by stimulating the transactivation domain of RelA/p65 in a manner that is dependent on IkappaB kinase beta activity and on the mitogen-activated protein kinase p38 (p38). Activation of RelA/p65 transactivation function requires serines 529 and 536, sites shown previously to be inducibly phosphorylated. Consistent with the requirement of p38 in the activation of NF-kappaB transcriptional function, expression of activated Akt induces p38 activity. Furthermore, the ability of IL-1beta to activate NF-kappaB is known to involve Akt, and we show here that IL-1beta induces p38 activity in manner dependent on Akt and IkappaB kinase activation. Interestingly, activated Akt and the transcriptional co-activators CBP/p300 synergize in the activation of the RelA/p65 transactivation domain, and this synergy is blocked by p38 inhibitors. These studies demonstrate that Akt, functioning through IkappaB kinase and p38, induces the transcription function of NF-kappaB by stimulating the RelA/p65 transactivation subunit of NF-kappaB.

Wnt-1 signaling inhibits apoptosis by activating beta-catenin/T cell factor-mediated transcription.

Wnt signaling plays a critical role in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of Wnt signaling, little is known regarding Wnt signaling modification of the cell death machinery. Given that numerous oncogenes transform cells by providing cell survival function, we hypothesized that Wnt signaling may inhibit apoptosis. Here, we report that cells expressing Wnt-1 were resistant to cancer therapy-mediated apoptosis. Wnt-1 signaling inhibited the cytochrome c release and the subsequent caspase-9 activation induced by chemotherapeutic drugs, including both vincristine and vinblastine. Furthermore, we found that Wnt-1-mediated cell survival was dependent on the activation of beta-catenin/T cell factor (Tcf) transcription. Inhibition of beta-catenin/Tcf transcription by expression of the dominant-negative mutant of Tcf-4 blocked Wnt-1-mediated cell survival and rendered cells sensitive to apoptotic stimuli. These results provide the first demonstration that Wnt-1 inhibits cancer therapy-mediated apoptosis and suggests that Wnt-1 may exhibit its oncogenic potential through a mechanism of anti-apoptosis.

Inhibition of NF-kappaB sensitizes non-small cell lung cancer cells to chemotherapy-induced apoptosis.

BACKGROUND: Most non-small cell lung cancers (NSCLC) are chemoresistant. Identification and modulation of chemoresistance cell-signaling pathways may sensitize NSCLC to chemotherapy and improve patient outcome. The purpose of this study was to determine if chemotherapy induces nuclear factor-kappa B (NF-kappaB) activation in NSCLC in vitro and whether inhibition of NF-kappaB would sensitize tumor cells to undergo chemotherapy-induced apoptosis. METHODS: Non-small cell lung cancer cells were treated with gemcitabine, harvested, and nuclear extracts analyzed for NF-kappaB DNA binding by electrophoretic mobility shift assays. Additionally, NSCLC cells that stably expressed a plasmid encoding the superrepressor IkappaBalpha protein (H157I) or a vector control (H157V) were generated. These cells were then treated with gemcitabine and apoptosis determined by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay. RESULTS: Chemotherapy induced NF-kappaB nuclear translocation and DNA binding in all NSCLC cell lines. H157I cells had enhanced cell death compared with H157V cells, suggesting that NF-kappaB is required for cell survival after chemotherapy. The observed cell death following the loss of NF-kappaB occurred by apoptosis. CONCLUSIONS: Inhibition of chemotherapy-induced NF-kappaB activation sensitizes NSCLC to chemotherapy-induced apoptosis in vitro. Novel treatment strategies for patients with advanced NSCLC may involve chemotherapy combined with inhibition of NF-kappaB-dependent cell-survival pathways.

NF-kappaB-induced loss of MyoD messenger RNA: possible role in muscle decay and cachexia.

MyoD regulates skeletal muscle differentiation (SMD) and is essential for repair of damaged tissue. The transcription factor nuclear factor kappa B (NF-kappaB) is activated by the cytokine tumor necrosis factor (TNF), a mediator of skeletal muscle wasting in cachexia. Here, the role of NF-kappaB in cytokine-induced muscle degeneration was explored. In differentiating C2C12 myocytes, TNF-induced activation of NF-kappaB inhibited SMD by suppressing MyoD mRNA at the posttranscriptional level. In contrast, in differentiated myotubes, TNF plus interferon-gamma (IFN-gamma) signaling was required for NF-kappaB-dependent down-regulation of MyoD and dysfunction of skeletal myofibers. MyoD mRNA was also down-regulated by TNF and IFN-gamma expression in mouse muscle in vivo. These data elucidate a possible mechanism that may underlie the skeletal muscle decay in cachexia.

The transcription factor NF-kappaB: control of oncogenesis and cancer therapy resistance.

Discovered in 1986 as a DNA binding activity that recognized the immunoglobulin light chain intronic enhancer, NF-kappaB has been studied intensively for its role in controlling expression of genes involved in immune and inflammatory function. However, more recently, NF-kappaB has been implicated in controlling cell growth and oncogenesis. The link between NF-kappaB and cancer stems, in part, from the fact that this transcription factor is capable of inducing gene products that control proliferative responses and that suppress apoptotic cascades, such as those induced by tumor necrosis factor (TNF), expression of oncoproteins, and genotoxic stress. This latter observation is likely to be important in developing new approaches aimed at improving the efficacy of cancer chemotherapy.

Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-kappaB.

It is well established that cell survival signals stimulated by growth factors, cytokines, and oncoproteins are initiated by phosphoinositide 3-kinase (PI3K)- and Akt-dependent signal transduction pathways. Oncogenic Ras, an upstream activator of Akt, requires NF-kappaB to initiate transformation, at least partially through the ability of NF-kappaB to suppress transformation-associated apoptosis. In this study, we show that oncogenic H-Ras requires PI3K and Akt to stimulate the transcriptional activity of NF-kappaB. Activated forms of H-Ras and MEKK stimulate signals that result in nuclear translocation and DNA binding of NF-kappaB as well as stimulation of the NF-kappaB transactivation potential. In contrast, activated PI3K or Akt stimulates NF-kappaB-dependent transcription by stimulating transactivation domain 1 of the p65 subunit rather than inducing NF-kappaB nuclear translocation via IkappaB degradation. Inhibition of IkappaB kinase (IKK), using an IKKbeta dominant negative protein, demonstrated that activated Akt requires IKK to efficiently stimulate the transactivation domain of the p65 subunit of NF-kappaB. Inhibition of endogenous Akt activity sensitized cells to H-Ras(V12)-induced apoptosis, which was associated with a loss of NF-kappaB transcriptional activity. Finally, Akt-transformed cells were shown to require NF-kappaB to suppress the ability of etoposide to induce apoptosis. Our work demonstrates that, unlike activated Ras, which can stimulate parallel pathways to activate both DNA binding and the transcriptional activity of NF-kappaB, Akt stimulates NF-kappaB predominantly by upregulating of the transactivation potential of p65.

A review of molecular recognition technologies for detection of biological threat agents.

The present review summarizes the state of the art in molecular recognition of biowarfare agents and other pathogens and emphasizes the advantages of using particular types of reagents for a given target (e.g. detection of bacteria using antibodies versus nucleic acid probes). It is difficult to draw firm conclusions as to type of biorecognition molecule to use for a given analyte. However, the detection method and reagents are generally target-driven and the user must decide on what level (genetic versus phenotypic) the detection should be performed. In general, nucleic acid-based detection is more specific and sensitive than immunological-based detection, while the latter is faster and more robust. This review also points out the challenges faced by military and civilian defense components in the rapid and accurate detection and identification of harmful agents in the field. Although new and improved sensors will continue to be developed, the more crucial need in any biosensor may be the molecular recognition component (e.g. antibody, aptamer, enzyme, nucleic acid, receptor, etc.). Improvements in the affinity, specificity and mass production of the molecular recognition components may ultimately dictate the success or failure of detection technologies in both a technical and commercial sense. Achieving the ultimate goal of giving the individual soldier on the battlefield or civilian responders to an urban biological attack or epidemic, a miniature, sensitive and accurate biosensor may depend as much on molecular biology and molecular engineering as on hardware engineering. Fortunately, as this review illustrates, a great deal of scientific attention has and is currently being given to the area of molecular recognition components. Highly sensitive and specific detection of pathogenic bacteria and viruses has increased with the proliferation of nucleic acid and immuno-based detection technologies. If recent scientific progress is a fair indicator, the future promises remarkable new developments in molecular recognition elements for use in biosensors with a vast array of applications.

Interleukin-10 signaling blocks inhibitor of kappaB kinase activity and nuclear factor kappaB DNA binding.

The transcription factor nuclear factor kappaB (NF-kappaB) coordinates the activation of numerous genes in response to pathogens and proinflammatory cytokines and is, therefore, pivotal in the development of acute and chronic inflammatory diseases. In its inactive state, NF-kappaB is constitutively present in the cytoplasm as a p50-p65 heterodimer bound to its inhibitory protein IkappaB. Proinflammatory cytokines, such as tumor necrosis factor (TNF), activate NF-kappaB by stimulating the activity of the IkappaB kinases (IKKs) which phosphorylate IkappaBalpha on serine residues 32 and 36, targeting it for rapid degradation by the 26 S proteasome. This enables the release and nuclear translocation of the NF-kappaB complex and activation of gene transcription. Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines which are known to be transcriptionally controlled by NF-kappaB. Conflicting data exists on the effects of IL-10 on TNF- and LPS-induced NF-kappaB activity in human monocytes and the molecular mechanisms involved have not been elucidated. In this study, we show that IL-10 functions to block NF-kappaB activity at two levels: 1) through the suppression of IKK activity and 2) through the inhibition of NF-kappaB DNA binding activity. This is the first evidence of an anti-inflammatory protein inhibiting IKK activity and demonstrates that IKK is a logical target for blocking inflammatory diseases.

NF-kappaB induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis.

Recent evidence indicates that the transcription factor NF-kappaB is a major effector of inducible antiapoptotic mechanisms. For example, it was shown that NF-kappaB activation suppresses the activation of caspase 8, the apical caspase in tumor necrosis factor (TNF) receptor family signaling cascades, through the transcriptional regulation of certain TRAF and IAP proteins. However, it was unknown whether NF-kappaB controls other key regulatory mechanisms in apoptosis. Here we show that NF-kappaB activation suppresses mitochondrial release of cytochrome c through the activation of the Bcl-2 family member A1/Bfl-1. The restoration of A1 in NF-kappaB null cells diminished TNF-induced apoptosis by reducing the release of proapoptotic cytochrome c from mitochondria. In addition, A1 potently inhibited etoposide-induced apoptosis by inhibiting the release of cytochrome c and by blocking caspase 3 activation. Our findings demonstrate that A1 is an important antiapoptotic gene controlled by NF-kappaB and establish that the prosurvival function of NF-kappaB can be manifested at multiple levels.

WT1 modulates apoptosis by transcriptionally upregulating the bcl-2 proto-oncogene.

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger transcription factor that has been demonstrated to negatively regulate several growth factor and cognate receptor genes. However, inconsistent with its tumor suppressor function, WT1 has also been demonstrated to be required to inhibit programmed cell death in vitro and in vivo. Moreover, anaplastic Wilms' tumors, which typically express wild-type WT1, display extreme resistance to chemotherapeutic agents that kill tumor cells through the induction of apoptosis. Although p53 mutations in anaplastic Wilms' tumors have been associated with chemoresistance, this event is believed to occur late during tumor progression. Therefore, since dysregulated WT1 expression occurs relatively early in Wilms' tumors, we hypothesized that WT1 was required to transcriptionally upregulate genes that provide a cell survival advantage to tumor cells. Here we demonstrate that sporadic Wilms' tumors coexpress WT1 and the anti-apoptotic Bcl-2 protein. Using rhabdoid cell lines overexpressing WT1, we show that WT1 activates the endogenous bcl-2 gene through a transcriptional mechanism. Transient transfections and electromobility shift assays demonstrate that WT1 positively stimulates the bcl-2 promoter through a direct interaction. Moreover, WT1 expressing cells displaying upregulated Bcl-2 were found to be resistant to apoptosis induced by staurosporine, vincristine and doxorubicine. These data suggest that in certain cellular contexts, WT1 exhibits oncogenic potential through the transcriptional upregulation of anti-apoptotic genes such as bcl-2.

NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation.

Tumor necrosis factor alpha (TNF-alpha) binding to the TNF receptor (TNFR) potentially initiates apoptosis and activates the transcription factor nuclear factor kappa B (NF-kappaB), which suppresses apoptosis by an unknown mechanism. The activation of NF-kappaB was found to block the activation of caspase-8. TRAF1 (TNFR-associated factor 1), TRAF2, and the inhibitor-of-apoptosis (IAP) proteins c-IAP1 and c-IAP2 were identified as gene targets of NF-kappaB transcriptional activity. In cells in which NF-kappaB was inactive, all of these proteins were required to fully suppress TNF-induced apoptosis, whereas c-IAP1 and c-IAP2 were sufficient to suppress etoposide-induced apoptosis. Thus, NF-kappaB activates a group of gene products that function cooperatively at the earliest checkpoint to suppress TNF-alpha-mediated apoptosis and that function more distally to suppress genotoxic agent-mediated apoptosis.

Requirement of NF-kappaB activation to suppress p53-independent apoptosis induced by oncogenic Ras.

The ras proto-oncogene is frequently mutated in human tumors and functions to chronically stimulate signal transduction cascades resulting in the synthesis or activation of specific transcription factors, including Ets, c-Myc, c-Jun, and nuclear factor kappa B (NF-kappaB). These Ras-responsive transcription factors are required for transformation, but the mechanisms by which these proteins facilitate oncogenesis have not been fully established. Oncogenic Ras was shown to initiate a p53-independent apoptotic response that was suppressed through the activation of NF-kappaB. These results provide an explanation for the requirement of NF-kappaB for Ras-mediated oncogenesis and provide evidence that Ras-transformed cells are susceptible to apoptosis even if they do not express the p53 tumor-suppressor gene product.

Induction of the transcription factor Sp1 during human cytomegalovirus infection mediates upregulation of the p65 and p105/p50 NF-kappaB promoters.

During human cytomegalovirus (HCMV) infection, the promoters for the classical NF-kappaB subunits (p65 and p105/p50) are transactivated. Previously, we demonstrated that the viral immediate-early (IE) proteins (IE1-72, IE2-55, and IE2-86) were involved in this upregulation. These viral factors alone, however, could not account for the entirety of the increased levels of transcription. Because one of the hallmarks of HCMV infection is the induction of cellular transcription factors, we hypothesized that one or more of these induced factors was also critical to the regulation of NF-kappaB during infection. Sp1 was one such factor that might be involved because p65 promoter activity was upregulated by Sp1 and both of the NF-kappaB subunit promoters are GC rich and contain Sp1 binding sites. Therefore, to detail the role that Sp1 plays in the regulation of NF-kappaB during infection, we initially examined Sp1 levels for changes during infection. HCMV infection resulted in increased Sp1 mRNA expression, protein levels, and DNA binding activity. Because both promoters were transactivated by Sp1, we reasoned that the upregulation of Sp1 played a role in p65 and p105/p50 promoter activity during infection. To address the specific role of Sp1 in p65 and p105/p50 promoter transactivation by HCMV, we mutated both promoters. These results demonstrated that the Sp1-specific DNA binding sites were involved in the virus-mediated transactivation. Last, to further dissect the role of HCMV in the Sp1-mediated induction of NF-kappaB, we examined the role that the viral IE genes played in Sp1 regulation. The IE gene products (IE1-72, IE2-55, and IE2-86) cooperated with Sp1 to increase promoter transactivation and physically interacted with Sp1. In addition, the IE2-86 product increased Sp1 DNA binding by possibly freeing up inactive Sp1. These data supported our hypothesis that Sp1 was involved in the upregulation of NF-kappaB during HCMV infection through the Sp1 binding sites in the p65 and p105/p50 promoters and additionally demonstrated a potential viral mechanism that might be responsible for the upregulation of Sp1 activity.

Basic fibroblast growth factor transcriptional autoregulation requires EGR-1.

Basic fibroblast growth factor (bFGF) is an important growth factor for neuroectoderm- and mesoderm-derived cells. In addition bFGF is an important angiogenic factor and appears to contribute to tumorigenesis. This is exemplified by the fact that bFGF is expressed in a large majority of human gliomas and that bFGF expression is critical for the growth and tumorigenesis of these cells. It has been shown previously that bFGF can induce its own expression through an increase in bFGF mRNA. In this report, we show that bFGF leads to its own synthesis through an autoregulated transcriptional response that requires the transcription factor Egr-1 (also known as Krox24, Zif268 and NGFI-A). Egr-1 binds to two DNA elements in the bFGF promoter and positively regulates transcription. Mutation of these sites blocks the ability of bFGF to transcriptionally regulate the bFGF promoter. These data indicate a mechanism to explain how bFGF functions to autoregulate its expression and demonstrate that Egr-1 is as an essential transcription factor in this process.