Paula C Fernandez

Universität Bern, Bern, BE, Switzerland

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Publications (17)116.99 Total impact

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    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.
    Cell cycle (Georgetown, Tex.) 11/2007; 6(20):2531-40. DOI:10.4161/cc.6.20.4807 · 5.01 Impact Factor
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    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-kappaB) activity. We found that this was mediated by recruitment of the multisubunit IkappaB 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.
    Science 12/2002; 298(5595):1033-6. DOI:10.1126/science.1075462 · 31.48 Impact Factor
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    ABSTRACT: The beta 2 subunit of the interleukin (IL)-12 receptor (IL-12R beta 2) has been shown to play an essential role in differentiation of T helper 1 (Th1) cells in the murine and human system, and antibodies raised against IL-12R beta 2 recognized this molecule on human Th1 but not Th2 cells. However, while the cytokines secreted by clones of murine cells allowed the definition of distinct T helper cell subsets, bovine clones with polarized Th1 and Th2 cytokine profiles were rarely found. This raised important questions about the regulation of immune responses in cattle. We therefore cloned bovine IL-12R beta2 (boIL-12R beta 2) DNA complementary to RNA (cDNA) from the start codon to the 3' end of the mRNA. Comparison of boIL-12R beta 2 cDNA with human and murine IL-12R beta 2 cDNA sequences revealed homologies of 85 and 78%, respectively. The deduced protein sequence showed the hallmark motifs of the cytokine receptor superfamily including the four conserved cysteine residues, the WSXWS motif and fibronectin domains in the extracellular part as well as a STAT4 binding site in the intracellular part of the molecule. Using real-time reverse transcription-polymerase chain reaction, upregulation of mRNA expression of this molecule could be demonstrated in cultured bovine lymph node cells stimulated with phytohemagglutinin. Furthermore, cells with upregulated boIL-12R beta 2 mRNA responded with enhanced expression of interferon gamma to treatment with interleukin 12.
    Gene 06/2002; 289(1-2):61-7. DOI:10.1016/S0378-1119(02)00464-X · 2.08 Impact Factor
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    ABSTRACT: Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-kappa B degradation and NF-kappa B activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-kappa B-mediated responses are regulated by selective combinations of NF-kappa B proteins as homo- or heterodimers and by distinct kappa B motifs. We characterised the NF-kappa B complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-kappa B/Rel family of proteins translocate to the nucleus of infected cells. Using two different kappa B oligonucleotides (kappa B-1 and kappa B-2), both containing the decameric consensus kappa B 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 kappa B-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 kappa B-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-kappa B activation pathways had no inhibitory effect on the activation of NF-kappa B DNA binding in TpM(803) T cells.
    Microbes and Infection 10/2000; 2(11):1311-20. · 2.73 Impact Factor
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    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.
    Microbes and Infection 09/2000; 2(11):1311-1320. DOI:10.1016/S1286-4579(00)01284-3 · 2.73 Impact Factor
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    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. © 2000 Éditions scientifiques et médicales Elsevier SAS parasite / signalling pathway / transcription factor
    Microbes and Infection 09/2000; · 2.73 Impact Factor
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    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.
    Cellular Microbiology 05/2000; 2(2):91-9. DOI:10.1046/j.1462-5822.2000.00045.x · 4.82 Impact Factor
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    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.
    Biological Chemistry 01/2000; 380(12):1383-94. DOI:10.1515/BC.1999.178 · 2.69 Impact Factor
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    ABSTRACT: The intracellular parasite Theileria parva transforms bovine T-lymphocytes, inducing uncontrolled proliferation. Upon infection, cells cease to require antigenic stimulation and exogenous growth factors to proliferate. Earlier studies have shown that pathways triggered via stimulation of the T-cell receptor are silent in transformed cells. This is reflected by a lack of phosphorylation of key signalling molecules and the fact that proliferation is not inhibited by immunosuppressants such as cyclosporin and ascomycin that target calcineurin. This suggests that the parasite bypasses the normal T-cells activation pathways to induce proliferation. Among the MAP-kinase pathways, ERK and p38 are silent, and only Jun N-terminal kinase is activated. This appears to suffice to induce constitutive activation of the transcription factor AP-1. More recently, it could be shown that the presence of the parasite in the host cell cytoplasm also induces constitutive activation of NF-kappaB, a transcription factor involved in proliferation and protection against apoptosis. Activation is effectuated by parasite-induced degradation of IkappaBs, the cytoplasmic inhibitors which sequester NF-kappaB in the cytoplasm. NF-kappaB activation is resistant to the antioxidant N-acetyl cysteine and a range of other reagents, suggesting that activation might occur in an unorthodox manner. Studies using inhibitors and dominant negative mutants demonstrate that the parasite activates a NF-kappaB-dependent anti-apoptotic mechanism that protects the transformed cell form spontaneous apoptosis and is essential for maintaining the transformed state of the parasitised cell.
    Veterinary Immunology and Immunopathology 12/1999; 72(1-2):95-100. DOI:10.1016/S0165-2427(99)00121-X · 1.75 Impact Factor
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    ABSTRACT: Nuclear factor-κB 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-κB 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-κB activation pathway, in primary and in transformed T cells. We show that NAC, contrary to its reported role as an NF-κB inhibitor, can actually enhance rather than inhibit IκB degradation and, most importantly, show that in all cases NAC exerts a dominant antagonistic effect on PDTC-mediated NF-κB inhibition. This was observed at the level of IκB degradation, NF-κB 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-κB activation pathways and shows that two compounds, previously thought to function through a common inhibitory mechanism, can also have antagonistic effects.
    Biological Chemistry 12/1999; 380(12):1383-1394. · 2.69 Impact Factor
  • Paula C. Fernandez, Dirk A.E. Dobbelaere
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    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.
    Biochemical and Biophysical Research Communications 10/1999; 263(1):63-7. DOI:10.1006/bbrc.1999.1312 · 2.28 Impact Factor
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    ABSTRACT: Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasite Theileria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-kappaB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-kappaB activation 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 transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-kappaB 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. Consequently, inhibition of NF-kappaB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-kappaB activation pathway, such as IkappaBalpha 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-kappaB activation is an essential step in maintaining the transformed phenotype of the infected cells.
    Proceedings of the National Academy of Sciences 07/1999; 96(13):7312-7. DOI:10.1073/pnas.96.13.7312 · 9.81 Impact Factor
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    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 I␬B␣ 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, I␬Bs, 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 I␬B by I␬B kinases (reviewed in ref. 34) targets I␬B 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 I␬B, resulting in constitutive nuclear translocation and transcriptional activity (36). Upon elimination of the parasite, I␬B 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.
    Proceedings of the National Academy of Sciences 07/1999; 96:7312-7317. · 9.81 Impact Factor
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    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).
    Cell Death and Differentiation 05/1999; 6(4):342-50. DOI:10.1038/sj.cdd.4400501 · 8.39 Impact Factor
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    ABSTRACT: The serine protease inhibitor N-a-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-kB activation in T. parva-transformed T-cells, with phosphorylation of IkBa and IkBb being inhibited with different kinetics. TPCK-mediated inhibition of IkB phosphor-ylation, NF-kB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-kB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-kB activation pathway(s).
    Cell death and differentiation 04/1999; · 8.39 Impact Factor
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    ABSTRACT: Infection of cattle with the protozoan Theileria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor kappaB (NFkappaB) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NFkappaB activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the IkappaB molecules which normally sequester NFkappaB in the cytoplasm and that continuous degradation requires viable parasites. Using T. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of IkappaBalpha. However, IkappaBalpha reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NFkappaB-mediated positive feedback loop which restores cytoplasmic IkappaBalpha. In contrast, T. parva mediated continuous degradation of IkappaBbeta resulting in persistently low cytoplasmic IkappaBbeta levels. Normal IkappaBbeta levels were only restored following T. parva killing, indicating that viable parasites are required for IkappaBbeta degradation. Treatment of T. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both IkappaB degradation and consequent enhanced expression of NFkappaB dependent genes. However treatment using the antioxidant N-acetylcysteine had no effect on either IkappaB levels or NFkappaB activation, indicating that the parasite subverts the normal IkappaB regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function.
    Proceedings of the National Academy of Sciences 12/1997; 94(23):12527-32. DOI:10.1073/pnas.94.23.12527 · 9.81 Impact Factor
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    ABSTRACT: Infection of cattle with the protozoan Thei-leria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Par-asitized cells exhibit autocrine growth characterized by per-sistent translocation of the transcriptional regulatory factor nuclear factor ␬B (NF␬B) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NF␬B activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the I␬B molecules which normally sequester NF␬B in the cyto-plasm and that continuous degradation requires viable par-asites. Using T. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteoly-sis of I␬B␣. However, I␬B␣ reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NF␬B-mediated positive feedback loop which restores cytoplasmic I␬B␣. In contrast, T. parva mediated continuous degradation of I␬B␤ resulting in persistently low cytoplasmic I␬B␤ levels. Normal I␬B␤ levels were only re-stored following T. parva killing, indicating that viable para-sites are required for I␬B␤ degradation. Treatment of T. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both I␬B degradation and consequent en-hanced expression of NF␬B dependent genes. However treat-ment using the antioxidant N-acetylcysteine had no effect on either I␬B levels or NF␬B activation, indicating that the parasite subverts the normal I␬B regulatory pathway down-stream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function. Pathogen-mediated lymphoproliferation involving nuclear fac-tor ␬B (NF␬B) activation is the defining feature of several diseases, most notably human T-lymphotropic virus, type I, induced adult T-cell leukemia (1–3), progression of bovine leukemia virus infection to B cell lymphoma (4, 5), and East Coast Fever caused by Theileria parva (6). T. parva is a protozoal pathogen of cattle that replicates in the cytoplasm of lymphocytes (7), inducing lymphoblastoid transformation re-sulting in clonal expansion of the infected cell (8, 9). These transformed cells, primarily T cells, disseminate to lymphoid and nonlymphoid tissues, including the lung, kidney, and, intestine, where they mimic the behavior of lymphoid tumors (10, 11). Remarkably unlike true lymphomas, T. parva-induced transformation can be reversed by killing the parasite (9, 10–12). This drug-induced reversal to normal lymphocyte regulation indicates that T. parva alone is sufficient and responsible for transformation. Our goal is to identify the mechanism used by T. parva to subvert normal signaling pathways with the expectation that this knowledge may pro-vide new approaches for disease control as well as increasing our understanding of normal T cell function. T. parva-infected lymphocytes are capable of continuous growth without addition of exogenous growth factors (13, 14). This autocrine growth is, at least partially, attributable to parasite-induced expression of both interleukin 2 (IL-2) and the ␣-chain of the IL-2 receptor (IL-2R␣) (9, 13–16). Treat-ment with the theilericidal drug BW720c (a protozoal-specific hydronaphthoquinone) results in down-regulation of IL-2 and IL-2R␣ expression and restoration of growth factor depen-dence (9, 12). Both IL-2 and IL-2R␣ are transcriptionally regulated by cis-acting ␬B enhancer elements that are, in turn, controlled by dimeric complexes of the NF␬B family of transcription factors (17, 18). Normally sequestered in the cytoplasm, NF␬B regulatory proteins, principally the p50͞Rel A heterodimer, are transiently translocated to the T cell nucleus following activation by antigen plus CD28 costimula-tion, mitogens, or cytokines, such as tumor necrosis factor-␣ (18, 19). Similarly, T. parva infection of T cells specifically induces nuclear translocation of NF␬B and binding to the ␬B enhancer (6). Importantly however, NF␬B translocation is persistent in the parasitized cell and requires viable T. parva (6). This indicates that the normal regulation of NF␬B is specifically altered by the protozoan. In T cells and in developing hematopoeitic organs, I␬B␣ is the major inhibitor of the p50͞RelA complex and sequesters the heterodimer in the cytoplasm via preferential binding to RelA and masking of the nuclear localization signals (18, 20–22). In response to antigen or cytokine activation, I␬B␣ is phosphorylated and targeted for degradation in the proteo-some (23–25). The freed p50͞Rel A complex is translocated to the nucleus where it binds the ␬B element resulting in tran-scriptional activation of genes required for proliferation, in-cluding IL-2 and IL-2R␣ (18, 19, 26). Importantly, I␬B␣ is also transcriptionally regulated by a ␬B enhancer and therefore subsequent increased I␬B␣ synthesis leads to re-established sequestration of p50͞RelA in the cytoplasm (18, 27, 28). This autoregulatory loop ensures that the NF␬B-induced gene expression is normally transient and tightly regulated. Persis-tent NF␬B activation is also associated with, and appears to require, degradation of a second inhibitor, I␬B␤ (29, 30). Unlike I␬B␣, I␬B␤ gene expression is not controlled by ␬B elements and restoration of normal cytoplasmic I␬B␤ levels
    Proceedings of the National Academy of Sciences 12/1997; 94:12527-12532. · 9.81 Impact Factor