[Show abstract][Hide abstract] ABSTRACT: The IkappaB kinase (IKK) complex controls processes such as inflammation, immune responses, cell survival and the proliferation of both normal and tumor cells. By activating NFkappaB, the IKK complex contributes to G1/S transition and first evidence has been presented that IKKalpha also regulates entry into mitosis. At what stage IKK is required and whether IKK also contributes to progression through mitosis and cytokinesis, however, has not yet been determined. In this study, we use BMS-345541, a potent allosteric small molecule inhibitor of IKK, to inhibit IKK specifically during G2 and during mitosis. We show that BMS-345541 affects several mitotic cell cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released from arrest in S-phase blocked the activation of Aurora A, B and C, Cdk1 activation and histone H3 phosphorylation. Additionally, treatment of the mitotic cells with BMS-345541 resulted in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 was also found to override the spindle checkpoint in nocodazole-arrested cells. In vitro kinase assays using BMS-345541 indicate that these effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. This study points towards a new potential role of IKK in cell cycle progression. Since deregulation of the cell cycle is one of the hallmarks of tumor formation and progression, the newly discovered level of BMS-345541 function could be useful for cell cycle control studies and may provide valuable clues for the design of future therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Parasites have evolved a plethora of mechanisms to ensure their propagation and evade antagonistic host responses. The intracellular
protozoan parasite Theileria is the only eukaryote known to induce uncontrolled host cell proliferation. Survival of Theileria-transformed leukocytes depends strictly on constitutive nuclear factor kappa B (NF-κB) activity. We found that this was mediated
by recruitment of the multisubunit IκB kinase (IKK) into large, activated foci on the parasite surface. IKK signalosome assembly
was specific for the transforming schizont stage of the parasite and was down-regulated upon differentiation into the nontransforming
merozoite stage. Our findings provide insights into IKK activation and how pathogens subvert host-cell signaling pathways.
[Show abstract][Hide abstract] ABSTRACT: Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-κB degradation and NF-κB activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-κB-mediated responses are regulated by selective combinations of NF-κB proteins as homo- or heterodimers and by distinct κB motifs. We characterised the NF-κB complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-κB/Rel family of proteins translocate to the nucleus of infected cells. Using two different κB oligonucleotides (κB-1 and κB-2), both containing the decameric consensus κB motif (GGGACTTTCC), clearly distinct patterns of DNA binding activities could be demonstrated in electrophoretic mobility shift assays. Supershift analysis and UV cross-linking assays showed that complexes binding to κB-1 consisted of p50, p65 and RelB homo and/or heterodimers. We could also detect an association of ATF-2 and c-Fos with one of the complexes. The HIV-derived κB-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-κB activation pathways had no inhibitory effect on the activation of NF-κB DNA binding in TpM(803) T cells.
No preview · Article · Sep 2000 · Microbes and Infection
[Show abstract][Hide abstract] ABSTRACT: The intracellular parasite Theileria parva infects and transforms bovine T-cells, inducing their uncontrolled proliferation and spread in non-lymphoid as well as lymphoid tissues. This parasite-induced transformation is the predominant factor contributing to the pathogenesis of a lymphoproliferative disease, called East Coast fever. T. parva-transformed cells become independent of antigenic stimulation or exogenous growth factors. A dissection of the signalling pathways that are activated in T. parva-infected cells shows that the parasite bypasses signalling pathways that normally emanate from the T-cell antigen receptor to induce continuous proliferation. This review concentrates on the influence of the parasite on the state of activation of the mitogen-activated protein kinase (MAPK), NF-kappaB and phosphoinositide-3-kinase (PI3-K) pathways in the host cell. Of the MAPKs, JNK, but not ERK or p38, is active, inducing constitutive activation of the transcription factors AP-1 and ATF-2. A crucial step in the transformation process is the persistent activation of the transcription factor NF-kappaB, which protects T. parva-transformed cells from spontaneous apoptosis accompanying the transformation process. Inhibitor studies also suggest an important role for the lipid kinase, PI-3K, in the continuous proliferation of T. parva-transformed lymphocytes.
Preview · Article · May 2000 · Cellular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Nuclear factor-kappaB regulates genes that control immune and inflammatory responses and are involved in the pathogenesis of several diseases, including AIDS and cancer. It has been proposed that reactive oxygen intermediates participate in NF-kappaB activation pathways, and compounds with putative antioxidant activity such as N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) have been used interchangeably to demonstrate this point. We examined their effects, separately and combined, on different stages of the NF-kappaB activation pathway, in primary and in transformed T cells. We show that NAC, contrary to its reported role as an NF-kappaB inhibitor, can actually enhance rather than inhibit IkappaB degradation and, most importantly, show that in all cases NAC exerts a dominant antagonistic effect on PDTC-mediated NF-kappaB inhibition. This was observed at the level of IkappaB degradation, NF-kappaB DNA binding, and HIV-LTR-driven reporter gene expression. NAC also counteracted growth arrest and apoptosis induced by dithiocarbamates. Antagonistic effects were further observed at the level of jun-NH2-terminal kinase, p38 and ATF-2 activation. Our findings argue against the widely accepted assumption that NAC inhibits all NF-kappaB activation pathways and shows that two compounds, previously thought to function through a common inhibitory mechanism, can also have antagonistic effects.
No preview · Article · Jan 2000 · Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Ceramide is a lipid second messenger which is generated in response to stimulation of a number of surface receptors, treatment with chemotherapeutic agents, or ionising radiation. Depending on the target cell, ceramide induces diverse biological responses including apoptosis, cell-cycle arrest, differentiation, and also proliferation. We studied the effect of ceramide on the degradation of IkappaB, the cytoplasmic inhibitor of the transcription factor NF-kappaB. We show that ceramide treatment results in reduced levels of phosphorylated IkappaBalpha and degradation of both IkappaBalpha and IkappaBbeta. Ceramide synergised with okadaic acid (OA), a compound which interferes with the protein phosphatase 2A-controlled component of the NF-kappaB activation pathway, enhancing OA-induced IkappaB degradation. Ceramide also synergised with phorbol 12-myristate 13-acetate, which mimics protein kinase C activation. Finally, we show that the synergistic effect of ceramide with OA or phorbol ester can be observed in primary lymph node T-cells as well as in transformed T-cells.
Preview · Article · Oct 1999 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Parasites have evolved a plethora of strate-gies to ensure their survival. The intracellular parasite Thei-leria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, induc-ing their continuous proliferation and rendering them meta-static. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-B, by inducing the constitutive degradation of its cyto-plasmic inhibitors. The biological significance of NF-B ac-tivation in T. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transfor-mation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-B activation plays a crucial role in the survival of T. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Conse-quently, inhibition of NF-B nuclear translocation and the expression of dominant negative mutant forms of components of the NF-B activation pathway, such as IB␣ or p65, prompt rapid apoptosis of T. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-B acti-vation is an essential step in maintaining the transformed phenotype of the infected cells. Theileria parva is an intracellular protozoan parasite that is transmitted by ticks and causes East Coast fever, a lympho-proliferative disorder of cattle in East and Central Africa (1). T. parva-induced T-cell transformation is the predominant mechanism underlying the pathogenesis of East Coast fever. Upon invasion by the parasite, T cells undergo lymphoblastoid transformation (2), become independent of antigen receptor stimulation (3, 4), and cease to require exogenous growth factors to proliferate (5, 6). T. parva-transformed T cells show many characteristics of tumor cells, including unlimited pro-liferation in vitro, clonal expansion, the formation of invasive tumors in nude mice, and lesions resembling multicentric lymphosarcoma (7, 8). Although the exact mode of transfor-mation has not yet been elucidated, a number of host cell kinases, such as Src-family kinases (9, 10), jun-NH 2 -terminal kinase (4, 11), and casein kinase II (12, 13), with important functions in the regulation of T-cell activation, proliferation, and transformation have been found to be activated in a parasite-dependent manner in T. parva-transformed T cells (9, 14, 15). A unique aspect of T. parva-induced transformation is that it is entirely reversible (5, 16). Continuous proliferation depends on the presence of T. parva in the host cell cytoplasm. When the parasite is eliminated by treatment with the specific theilericidal drug BW720c, cells revert to a resting phenotype over a period of 3–4 days (5) and eventually undergo apopto-sis (10). Apoptosis is a widespread mechanism that is central to the maintenance of cellular homeostasis in all tissues, including the immune system (17). Apoptosis, or the lack of apoptosis, contributes to the pathogenesis of a number of diseases, including viral infection, autoimmune disease, and, in partic-ular, cancer (18, 19). In the latter context, evidence has been accumulating that oncogenesis requires an antiapoptotic func-tion that protects transformed cells from programmed cell death (20). The transcription factor NF-B, which regulates the expression of a diverse set of genes involved in immune function, differentiation, and proliferation (21), also has been shown to contribute to cell survival by conveying protection against cell death, induced by a range of different apoptotic stimuli (22–28). It recently has been shown that this protection is conveyed by the induction of genes that encode different inhibitor of apoptosis proteins (29–32), which inhibit members of the caspase family of cell-death proteases (33). NF-B is regulated by cytoplasmic inhibitor proteins, IBs, which sequester NF-B in the cytoplasm, preventing its trans-location to the nucleus and induction of B-dependent gene expression. Signal-induced phosphorylation of IB by IB kinases (reviewed in ref. 34) targets IB to a proteasomal degradation pathway, resulting in NF-B release, nuclear translocation, and activation. In earlier work, it was demon-strated that transformation of T. parva-infected T cells is accompanied by high levels of NF-B in the nucleus (35). More recently, it was shown that this transformation involves con-tinuous parasite-dependent degradation of IB, resulting in constitutive nuclear translocation and transcriptional activity (36). Upon elimination of the parasite, IB degradation and NF-B nuclear translocation are down-regulated (35, 36) and the expression of NF-B-responsive genes such as the IL-2 and IL-2R genes (5, 37–39) is arrested. Given that oncogenesis in tumor cells requires a functional antiapoptotic mechanism (18, 19, 40–42) and that NF-B has been shown to suppress apoptosis induced by a range of stimuli, we investigated the specific role of parasite-induced NF-B activation in the survival of T. parva-transformed T cells. We demonstrate that a stimulus to undergo spontaneous apoptosis is activated in T. parva-transformed T cells that is blocked, however, by activated NF-B. Thus, in addition to inducing the expression of genes that contribute to prolifera-tion (5, 37–39), permanently activated NF-B is also critically required for the survival of T. parva-transformed T cells.
Full-text · Article · Jul 1999 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The serine protease inhibitor N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) can interfere with cell-cycle progression and has also been shown either to protect cells from apoptosis or to induce apoptosis. We tested the effect of TPCK on two transformed T-cell lines. Both Jurkat T-cells and Theileria parva-transformed T-cells were shown to be highly sensitive to TPCK-induced growth arrest and apoptosis. Surprisingly, we found that the thiol antioxidant, N-acetylcysteine (NAC), as well as L- or D-cysteine blocked TPCK-induced growth arrest and apoptosis. TPCK inhibited constitutive NF-kappaB activation in T. parva-transformed T-cells, with phosphorylation of IkappaBalpha and IkappaBbeta being inhibited with different kinetics. TPCK-mediated inhibition of IkappaB phosphorylation, NF-kappaB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-kappaB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-kappaB activation pathway(s).
Full-text · Article · May 1999 · Cell Death and Differentiation