The transcription factor NF-B is one of the key regulators of genes involved in the immune and inflammatory response (for review, see Ref. 2). In mammalian cells, NF-B is composed of a homo-or heterodimer of various DNA-binding subunits. Five different DNA-binding subunits share a N-terminal homology domain, which confers DNA binding, dimerization, nuclear translocation, and interaction with the inhibitory IB proteins (for review, see Refs. 3 and 4). In most cell types these proteins sequester NF-B, which is frequently a heterodimer of the p50 and p65 (RelA) subunits, in the cytoplasm by masking their nuclear localization sequence. Constitutive NF-B activity in the cell nucleus can only be detected in certain neurons, some cells of the monocyte/macrophage lineage and B cells (for review, see Refs. 5 and 6

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To test whether the behaviour of transcription factor NF-kB as a promoter or antagonist of apoptosis depends on the apoptotic stimulus, we determined the influence of NF-kB on cell killing elicited by a variety of inducers within a given cell type. Inhibition of NF-kB by genetic and pharmacological approaches rendered HeLa cells more susceptible to TNF-a-induced cell killing, but protected them almost completely from H 2 O 2 -and pervanadate-induced apoptosis. TNF-a was unable to protect HeLa from H 2 O 2 -and pervanadate-induced apoptosis and further enhanced the cytotoxicity induced by these two adverse agents. Supernatants from HeLa cells stably overexpressing a transdominant negative form of IkB-a selectively increased the cytotoxicity of TNF-a for HeLa cells, suggesting that the enhanced susceptibility of these cells can be attributed to one or more secretable factors. Supershift experiments showed that the various apoptotic stimuli induced the same subset of DNA-binding subunits. Therefore, the nature of the signals elicited by the respective death inducers determines whether NF-kB induction leads to apoptosis or survival, suggesting that the manipulation of NF-kB activity may provide a new approach to adjuvant therapy in cancer treatment.Keywords: apoptosis; NF-kB; cancer therapy; TNF-a.In most cell types, the DNA-binding dimer of nuclear factor kB (NF-kB) is retained in the cytoplasm by interaction with an inhibitory protein, called inhibitor of NF-kB (IkB) [1]. A wide variety of stimuli including tumor necrosis factor a (TNF-a), interleukin-1 (IL-1) and T-cell activation lead to phosphorylation of the two major forms of IkB proteins, termed IkB-a and IkB-b. The inducible phosphorylation at serines 32 and 36 of IkB-a is a signal for subsequent ubiquitinylation and degradation by the 26S subunit of the proteasome [2,3]. There is recent evidence for the existence of alternative NF-kB activation pathways. UV radiation leads to the phosphorylation-independent degradation of 5], and reoxygenation-induced tyrosine phosphorylation of IkB-a allows its association with the regulatory subunit of phosphatidylinositol 3 H -kinase, thereby sequestering the inhibitory subunit from NF-kB [6]. Besides its well-established role for the immune response, there is growing evidence for an important function of NF-kB in cell proliferation [7,8] and apoptosis [9]. Anti-apoptotic activities of NF-kB are observed following some apoptotic stimuli, including the cytokine TNF-a, ionizing radiation and the cancer chemotherapeutic agent daunorubicine [10±13]. This protective effect of NF-kB on TNF-a-induced apoptosis is also seen in tumor-bearing mice [14]. In addition, p53-independent apoptosis induced by oncogenic Ras is suppressed by NF-kB activation [15]. On the other hand, there is ample evidence for apoptosispromoting functions of NF-kB. CD4 1 /CD8 1 thymocytes from mice overexpressing a transdominant negative form of IkB-a are resistant to activation-induced cell death [16]. The execution of NF-kB-dependent apoptosis, however,...

Infection with high-risk human papillomaviruses (HPV) can lead to the development of cervical carcinomas. This process critically depends on the virus-encoded E6 and E7 oncoproteins, which stimulate proliferation by manipulating the function of a variety of host key regulatory proteins. Here we show that both viral proteins dose-dependently interfere with the transcriptional activity of NF-B. A variety of experimental approaches revealed that a fraction of the E7 proteins is found in association with the IB kinase complex and attenuates induced kinase activity of IB kinase ␣ (IKK␣) and IKK␤, thus resulting in impaired IB␣ phosphorylation and degradation. Indirect immunofluorescence shows that E7 impairs TNF␣-induced nuclear translocation of NF-B, thus preventing NF-B from binding to its cognate DNA. While E7 obviates IKK activation in the cytoplasm, the E6 protein reduces NF-B p65-dependent transcriptional activity within the nucleus. We suggest that HPV oncogene-mediated suppression of NF-B activity contributes to HPV escape from the immune system. HPVs1 are small DNA viruses, and specific high-risk types such as the HPV type 16 (HPV16) or HPV18 are causative agents of some forms of anogenital and oral cancers (1). HPV16 encodes six early proteins including the major oncoproteins E6 and E7. Both proteins play a central role in the induction of benign proliferation and malignant transformation (2), and at least the persistence of E7 is necessary to maintain the transformed phenotype (3). These two oncoproteins are selectively and continuously expressed in HPV-induced tumors and manipulate cell proliferation upon physical and functional interaction with several master cell cycle regulators (4). E6 binds to p53 (5) and causes its ubiquitin-dependent degradation (6), thereby interfering with p53 functions in cell cycle control and apoptosis. In addition, the E6 protein binds to the protein kinase PKN (7) and other regulators including interferon regulatory factor 3 (8) and the proapoptotic Bak protein (9). The E7 protein interacts with so-called "pocket proteins" such as the retinoblastoma protein pRb, p107, and p130 (10), resulting in their enhanced phosphorylation and degradation (11). pRb destruction results in the release of E2F family transcription factors and subsequent activation of genes promoting cell proliferation (12). But the stimulatory effects of E7 on cell proliferation depends not only on its association with pRb (13, 14), because E7 targets the function of a plethora of regulators including cyclin E (15), acid alpha-glucosidase (16), and M2 pyruvate kinase (17). E7 also interferes with the activity of a variety of transcription factors such as AP-1 (18), interferon regulatory factor-1 (19), fork head domain transcription factor MPP2 (20), and TATA-box-binding protein (21). This multiplicity of interaction partners and additional levels of functional E7 regulation by phosphorylations (22), protein stability (23), and the oligomerization state (24) allow a highly complex and sophisticated manipulatio...

Exposure of T cells to the macrophage products hydrogen peroxide (HP) or l-lactate (LAC) was previously shown to enhance IL-2 production and to modulate glutathione (GSH) status. We now found that 50 μM HP and 30 mM LAC enhanced strongly the transcription from the IL-2 promoter in Jurkat T cells after stimulation with anti-CD28 together with or without anti-CD3 but not with anti-CD3 Abs alone. Therefore, we used anti-CD3 plus anti-CD28-stimulated cells to investigate the effect of the GSH reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on the signal cascade. BCNU enhanced the transcription to a similar extent as HP or LAC. Lowering the intracellular GSH/GSH disulfide ratio by BCNU, HP, or NO resulted in all cases in the fulminant enhancement of Jun-N-terminal kinase and p38 mitogen-activated protein kinase but not extracellular signal-regulated kinase 1/2. Jun-N-terminal kinase and NF-κB activation was enhanced through pathways involving Rac, Vav1, PKCΘ, p56lck, p59fyn, and IκB kinases. In a cell-free system, the autophosphorylation of rFyn was stimulated by GSH disulfide but not by HP. These findings suggest that the oxidation of the cellular thiol pool may play a role as an amplifying mechanism for TCR/CD3 signals in immune responses.

The phosphorylation of IκB by the multiprotein IκB kinase complex (IKC) precedes the activation of transcription factor NF-κB, a key regulator of the inflammatory response. Here we identified the mixed-lineage group kinase 3 (MLK3) as an activator of NF-κB. Expression of the wild-type form of this mitogen-activated protein kinase kinase kinase (MAPKKK) induced nuclear immigration, DNA binding, and transcriptional activity of NF-κB. MLK3 directly phosphorylated and thus activated IκB kinase alpha (IKKα) and IKKβ, revealing its function as an IκB kinase kinase (IKKK). MLK3 cooperated with the other two IKKKs, MEKK1 and NF-κB-inducing kinase, in the induction of IKK activity. MLK3 bound to components of the IKC in vivo. This protein-protein interaction was dependent on the central leucine zipper region of MLK3. A kinase-deficient version of MLK3 strongly impaired NF-κB-dependent transcription induced by T-cell costimulation but not in response to tumor necrosis factor alpha or interleukin-1. Accordingly, endogenous MLK3 was phosphorylated and activated by T-cell costimulation but not by treatment of cells with tumor necrosis factor alpha or interleukin-1. A dominant negative version of MLK3 inhibited NF-κB- and CD28RE/AP-dependent transcription elicited by the Rho family GTPases Rac and Cdc42, thereby providing a novel link between these GTPases and the IKC.

In this study we identified tyrosine-phosphorylatedSolely triggering the T-cell receptor (TCR) 1 without stimulation of accessory receptors leads to a state of unresponsiveness termed anergy (1). Full activation of T-lymphocytes necessarily requires two signals (2). The first signal is provided by the interaction of major histocompatibility complex molecules loaded with processed foreign antigens on antigen-presenting cells with the specific TCR⅐CD3⅐CD4 complex. The second signal is mediated by the occupancy of auxiliary receptors such as CD28. The two signaling pathways derived either from TCR or CD28 merge and synergistically stimulate the activity of JNK, NF-B, and the expression of various target genes including IL-2, which promotes the proliferation and differentiation of Tcells and is therefore required for the full immune response (3). Costimulation also induces an actin/myosin-dependent, directional transport of proteins including the TCR and lipid domains to a cap structure, termed the immunological synapse (4). One of the earliest events in TCR signaling is the activation of cytoplasmic protein tyrosine kinases, including members of the Src (Lck and Fyn) and Syk (ZAP70 and Syk) families (5). Induced tyrosine phosphorylation of target proteins allows the formation of multiprotein complexes at the inner leaflet of the cell membrane. Membrane anchorage is mediated by transmembrane adaptor proteins including linker for activation of T-cells (LAT) (6), which serve as docking ports for the formation of multiprotein complexes. This complex, which we refer to as T-cell activation signaling complex (TASC), contains other crucial signaling molecules such as PLC␥, phosphatidylinositol 3-kinase, and the adaptor protein SLP76 (7). The TASC propagates the signals and links them to multiple downstream signaling pathways. Tyrosine-phosphorylated LAT binds to PLC␥ which then cleaves phosphatidylinositol diphosphate thus generating inositol 1,4,5-trisphosphate and diacylglycerol. Whereas diacylglycerol mediates activation of protein kinase C family members (8), inositol 1,4,5-trisphosphate mobilizes Ca 2ϩ from intracellular stores. The Ca 2ϩ -mediated activation of the serine phosphatase calcineurin stimulates the nuclear entry of transcription factor NF-ATc (9).Among the substrates for protein tyrosine kinases is also the Vav1 protein, which is exclusively expressed in hematopoietic cells. The 95-kDa product of the Vav1 proto-oncogene displays a unique arrangement of signaling motifs including a calponin homology domain, an acidic domain, a DBL homology (DH) domain, a pleckstrin homology (PH) domain, a cysteine-rich domain (CR), a SH2 domain flanked by two proline-binding SH3 domains, and a bipartite putative nuclear localization signal (10). In vitro experiments show that Vav1, once activated by phosphatidylinositol-3,4,5-triphosphate binding and Lck phosphorylation, stimulates the GDP/GTP exchange activity of Rac (11,12). Therefore, Vav1 is a guanine nucleotide exchange factor (GEF) with selectivity for the Rho famil...

NF-kB is an inducible transcription factor, which is regulated by interaction with inhibitory IkB proteins. Previous studies linked the activity of NF-kB to the proliferative state of the cell. Here we have analysed the function of NF-kB in the cell cycle. Inhibition of NF-kB in HeLa cells by stable overexpression of a transdominant negative IkB-a protein reduced cell growth. A kinetic analysis of the cell cycle revealed a retarded G1/S transition. The IkB-a overexpressing cell clones showed a decreased percentage of cells in the S phase and an impaired incorporation of bromodeoxyuridine (BrdU). The amounts of cyclins A, B1, D1, D3, and E were unchanged, but the G1-speci®c proteins cyclin D2 and cdk2 were strongly elevated in the IkB-a overexpressing cell clones. These cell clones also displayed an increase in cyclin D1-dependent kinase activity, pointing to a cell cycle arrest at the late G1 phase. IkB-a overexpression crosstalked to cell cycle checkpoints via a reduction of transcription factor p53 and elevation of p21 WAF . Surprisingly, the IkB-a overexpressing cells showed an enrichment of c-Myc in the nucleoli, although the total amount of c-Myc protein was unchanged. These experiments identify an important contribution of the NF-kB/IkB system for the growth of HeLa cells.